Compositions and methods for cell immunotherapy

Abstract

Provided are methods for treating a subject of a solid tumor expressing GPC3 comprising administering to the subject an anti-GPC3 chimeric antigen receptor immunoresponsive cell wherein the administering is performed after or concurrently with subjecting the subject to a lymphopenia treatment. A kit for use in the method is also provided.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202510118979.0 (22) Application Date 2017.04.21 (66) Domestic Priority Data 201610256568.9 2016.04.22 CN (62) Divisional Application Data 201780038997.1 2017.04.21 (71) Applicant Craig Medical Limited Address Room 12, 20th Floor, Ho King Commercial Centre, 2-16 Garden Street, Mong Kok, Kowloon, Hong Kong (72) Inventors Li Zonghai, Gao Huiping, Wang Peng, Jiang Hua, Wang Huamao (74) Patent Agency Beijing Caihe Law Firm 11688 Patent Attorney Yan Sangtian, Li Min (51) Int.Cl. A61K 40 / 11 (2025.01) A61K 40 / 15(2025.01) A61K 40 / 31(2025.01) A61K 40 / 42(2025.01) A61P 35 / 00(2006.01) C07K 16 / 30(2006.01) A61K 45 / 06(2006.01) A61K 31 / 7048(2006.01) A61K 31 / 7068(2006.01) A61K 31 / 519(2006.01) A61K 31 / 475(2006.01) A61K 31 / 704(2006.01) (54) Title of Invention Composition and Method for Cellular Immunotherapy (57) Abstract A method for treating a subject with a solid tumor expressing GPC3 is provided, comprising administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or concurrently with lymphopenia treatment of the subject. A kit for use in the method is also provided. Claims 2 pages Description 55 pages Sequence Listing (electronic publication) Drawings 16 pages CN 121102467 A 2025.12.12 CN 1 21 10 24 67 A 1. A method for treating a subject with a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3), the method comprising administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or concurrently with lymphopenia treatment of the subject, wherein the lymphopenia treatment comprises administering cyclophosphamide and fludarabine to the subject. 2. The method of claim 1, wherein the immune response cells are NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells). 3. The method of claim 2, wherein the anti-GPC3-CAR is administered to the subject.4. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is at least about 5 × 10⁴ cells / kg. 5. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is from about 5 × 10⁴ to about 1 × 10¹² cells / kg. 6. The method of claim 1, wherein the administration effectively reduces the tumor size by at least 30%, the tumor size being measured by computed tomography (CT) scan. 7. The method of claim 1, wherein the administration effectively stabilizes the tumor size, the stabilized tumor size being a change of less than 10% from a baseline measurement of the tumor lesion diameter as measured by computed tomography (CT) scan. 8. The method of claim 2, wherein the combination of administration of anti-GPC3-CAR T cells and lymphopenia treatment synergistically extends the median survival of the subject by at least about 6 months compared to administration of the anti-GPC3-CAR T cells alone. 9. The method of claim 1, wherein the solid tumor is liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell carcinoma, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer. 10. The method of claim 1, wherein the anti-GPC3 chimeric antigen receptor comprises an antigen-binding unit that specifically binds to the C-terminus of GPC3. 11. The method of claim 10, wherein the antigen-binding unit comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. 12. The method of claim 1, wherein the anti-GPC3-CAR comprises a sequence selected from SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30. 13. The method of claim 1, wherein the anti-GPC3-CAR comprises a sequence selected from SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:30.28. The sequence shown in SEQ ID NO 29 or SEQ ID NO 30. 14. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least two intracellular signal transduction domains. 15. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least three intracellular signal transduction domains. 16. The method of claim 14, wherein the intracellular signal transduction domain is selected from the signal transduction domains of CD3, CD28, 4-1BB, OX40, DAP10, or ICOS. Claims 1 / 2 page 2 CN 121102467 A 17. The method of claim 1, wherein the lymphopenia treatment comprises reducing the amount of regulatory T cells in the subject. 18. The method of claim 17, wherein reducing the amount of regulatory T cells comprises reducing at least about 30% of the regulatory T cells, the amount of regulatory T cells being measured by flow cytometry analysis of circulating CD4+ and CD25+ cells in the subject. 19. The method of claim 1, wherein the lymphopenia treatment further comprises administering radiation or a biological agent to the subject. 20. The method of claim 1, wherein the lymphopenia treatment further comprises administering other chemotherapy to the subject. 21. The method of claim 20, wherein administering other chemotherapy to the subject comprises administering a chemotherapeutic agent selected from etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof. 22. The method of claim 21, wherein the chemotherapeutic agent is administered to the subject at least once prior to the administration of the anti-GPC3-CAR T cells. 23. The method of claim 1, wherein the lymphopenia treatment reduces the number of lymphocytes by at least about 20%, the number of lymphocytes being measured by complete blood cell count (CBC) analysis. 24. The method of claim 2, further comprising administering the anti-GPC3-CAR T cells to the subject a second time. 25. The method of claim 1, wherein the subject suffers from a refractory, persistent, or progressive disease. 26. The method of claim 2, wherein the anti-GPC3-CAR T cells are autologous or allogeneic relative to the subject. 27. The method of claim 1, further comprising administering at least one immunostimulant to the subject concurrently with or after the administration of the anti-GPC3 chimeric antigen receptor immune response cells. 28. The method of claim 27, wherein the immunostimulant is selected from aldehyde interleukin (IL-2), IL-3, IL-6, IL-11, GM-CSF, and any combination thereof.29. The method of claim 19, wherein the biological agent is an antibody targeting an antigen expressed on lymphocytes. Claims 2 / 2 Page 3 CN 121102467 A Compositions and methods for cell immunotherapy Cross-reference

[0001] This application claims the benefit of Chinese Patent Application No. CN201610256568.9, filed April 22, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] This application is a divisional application of Patent Application No. CN201780038997.1, filed April 21, 2017, entitled “Compositions and methods for cell immunotherapy”. Background Art

[0003] Cancer has a significant social impact worldwide. In 2016, an estimated 1,685,210 new cancer cases were diagnosed in the United States alone, and 595,690 people were expected to die from the disease. According to the Journal of Oncology Practice (Erikson 2007), by 2020, approximately 1 in 19 of the 18.2 million Americans would be a cancer patient or cancer survivor, an increase from 1 in 26 Americans in 2005.

[0004] Chimeric antigen receptors (CARs) are recombinant receptors for antigens that redirect the specificity and function of T cells and other immune cells to a single molecule. The use of CARs in cancer immunotherapy can bypass the barriers of active immunity and rapidly generate tumor-targeting T cells. Once expressed in cells, CAR-modified cells can exert immediate and long-term effects in subjects.

[0005] Chimeric antigen receptor (CAR) T-cell therapy, which edits the T cells of cancer patients to recognize their tumors, has shown efficacy in treating blood cancers. In recent clinical trials, CAR T-cell therapy has significantly improved treatment outcomes for patients with advanced, otherwise incurable blood cancers such as leukemia and lymphoma. In contrast, CAR T-cell therapy for solid tumors faces a unique set of challenges. The challenges include identifying antigens that clearly distinguish tumors from normal tissues, and establishing effective killing of tumor cells within the tumor to reduce tumor size. Summary of the Invention

[0006] There is an urgent need for alternative and effective treatments for a variety of solid tumors. This invention addresses this need and also provides related advantages. Therefore, this invention discloses a method for treating a subject exhibiting a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3). In some cases, the method may include administering anti-GPC3 chimeric antigen receptor immune response cells to the subject. In some cases, administration may be performed on a subject receiving lymphopenia treatment.This can be performed subsequently or simultaneously. In some cases, the immune response cells may be NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells). In some cases, administration of anti-GPC3-CAR T cells to the subject may be performed after the subject has undergone lymphopenic therapy. In some cases, the amount of GPC3-CAR T cells that can be administered to the subject is at least about 5 × 10⁴ cells / kg. In some cases, the amount of GPC3-CAR T cells administered to the subject is about 5 × 10⁴ to about 1 × 10¹² cells / kg. Administration can effectively reduce tumor size by at least 30%, as measured by computed tomography (CT) scans. In some cases, administration can effectively stabilize tumor size, which is defined as a change of less than 10% from the baseline measurement of the tumor lesion diameter as measured by computed tomography (CT) scans. In some cases, administration of anti-GPC3-CAR T cells in combination with lymphopenic therapy can synergistically extend the median survival of the subject by at least about 6 months compared to administration of anti-GPC3-CAR T cells alone. In some cases, solid tumors can be liver cancer, stomach cancer, thyroid cancer (e.g., thyroid tumors), lung cancer, breast cancer, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell cancer, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer. In some cases, anti-GPC3 chimeric antigen receptors may include antigen-binding units that can specifically bind to the C-terminus of GPC3. In some cases, the antigen-binding unit may contain a sequence that exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some cases, anti-GPC3-CAR may contain SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ IDSequences NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30 exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity. Anti-GPC3-CAR may comprise sequences exhibiting at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with any of SEQ ID NOs 28 to 30. In some cases, anti-GPC3-CAR T cells may contain at least two intracellular signaling domains. In some cases, anti-GPC3-CAR T cells may contain at least three intracellular signaling domains. The intracellular signaling domains may be selected from signaling domains derived from CD3, CD28, 4-1BB, OX40, DAP10, or ICOS. In some cases, lymphopenic treatment may include reducing the number of regulatory T cells in the subject. Reducing the number of regulatory T cells may include reducing at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the regulatory T cells, the number of which is measured by flow cytometry analysis of circulating CD4+ and CD25+ cells in the subject. In some cases, lymphopenic treatment may include administration of radiation or biological agents to the subject. Lymphopenic treatment may also include administration of chemotherapy to the subject. Administering chemotherapy to a subject may include administering a chemotherapeutic agent selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof. In some cases, the subject may be given at least one chemotherapeutic agent prior to administration of anti-GPC3-CAR T cells. In some cases, lymphopenic treatment may reduce the number of lymphocytes by at least about 20%, the number of lymphocytes being measured by complete blood count (CBC) analysis.

[0007] This document may also disclose a method further comprising administering a second dose of anti-GPC3-CAR T cells to the subject. In some cases, the subject may have a refractory, persistent, or progressive disease. The anti-GPC3-CAR T cells may be autologous or allogeneic relative to the subject.

[0008] This document may also disclose a method further comprising administering at least one immunostimulant to the subject concurrently with or after administration of anti-GPC3 chimeric antigen receptor immune response cells. The immunostimulant may be selected from interleukin-2 (IL-2).IL-3, IL-6, IL-11, GM-CSF, and any combination thereof. The biological agent may be an antibody against an antigen expressed on lymphocytes.

[0009] This document discloses a kit for administering anti-GPC3-CAR immune-response cells to a subject exhibiting a solid tumor, comprising: an effective amount of anti-GPC3-CAR immune-response cells; a chemotherapeutic agent effective in reducing the lymphocytes present in the subject; and instructions for administering the anti-GPC3-CAR immune-response cells to the subject after or simultaneously with the chemotherapeutic agent. In some cases, the anti-GPC3-CAR immune-response cells may be NK cells or T cells (anti-GPC3-CAR T cells). The chemotherapeutic agent may be selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof. The kit may further comprise about 60 mg / kg to about 80 mg / kg of cyclophosphamide or about 25 mg / m² to about 35 mg / m² of fludarabine formulated for administration to a subject in need. In some cases, the kit may contain approximately 1 x 10⁴ cells to approximately 1 x 10¹² anti-GPC3-CAR T cells. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells at least 12 hours after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3-CAR T cells at least 24 hours after chemotherapy. Anti-GPC3-CAR T cells may be formulated for intravenous injection. Anti-GPC3-CAR T cells may be formulated for intra-arterial injection into the liver of a subject who may contain a solid tumor.

[0010] Specifically, this application provides the following technical solutions:

[0011] 1. A method for treating a subject with a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3), the method comprising administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or simultaneously with lymphopenia treatment of the subject.

[0012] 2. The method of claim 1, wherein the immune response cells are NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells).

[0013] 3. The method of claim 2, wherein the administration of the anti-GPC3-CAR T cells to the subject is performed after the subject has received lymphopenia treatment.

[0014] 4. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is at least about 5 × 10⁴ cells / kg.

[0015] 5. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is about 5 × 10⁴ to about 1 × 10¹² cells / kg.

[0016] 6. The method of claim 1, wherein the administration effectively reduces the tumor size by at least 30%, the tumor size being measured by computed tomography (CT) scan.

[0017] 7. The method of claim 1, wherein the administration effectively stabilizes the tumor size, the stabilized tumor size being a change of less than 10% from a baseline measurement of the tumor lesion diameter as measured by computed tomography (CT) scan.

[0018] 8. The method of claim 2, wherein the combination of administration of anti-GPC3-CAR T cells and lymphopenia treatment synergistically extends the median survival of the subject by at least about 6 months compared to administration of the anti-GPC3-CAR T cells alone.

[0019] 9. The method of claim 1, wherein the solid tumor is liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell cancer, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer.

[0020] 10. The method of claim 1, wherein the anti-GPC3 chimeric antigen receptor comprises an antigen-binding unit that specifically binds to the C-terminus of GPC3.

[0021] 11. The method of claim 10, wherein the antigen-binding unit comprises a sequence exhibiting at least 90% sequence homology to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0022] 12. The method according to claim 1, wherein the anti-GPC3-CAR comprises a sequence exhibiting at least 90% sequence identity with SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.

[0023] 13. The method according to claim 1, wherein the anti-GPC3-CAR comprises a sequence exhibiting at least 90% sequence identity with SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30.30 sequences exhibiting at least 90% sequence identity.

[0024] 14. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least two intracellular signal transduction domains. Specification 3 / 55 page 6 CN 121102467 A

[0025] 15. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least three intracellular signal transduction domains.

[0026] 16. The method of claim 14 or 15, wherein the intracellular signal transduction domain is selected from: CD3, CD28, 4-1BB, OX40, DAP10, or ICOS signal transduction domains.

[0027] 17. The method of claim 1, wherein the lymphopenia treatment comprises reducing the amount of regulatory T cells in the subject.

[0028] 18. The method of claim 17, wherein the reduction in the amount of regulatory T cells comprises reducing the number of regulatory T cells by at least about 30%, the amount of regulatory T cells being measured by flow cytometry analysis of circulating CD4+ and CD25+ cells in the subject.

[0029] 19. The method of claim 1, wherein the lymphopenia treatment further comprises administering radiation or a biological agent to the subject.

[0030] 20. The method of claim 1, wherein the lymphopenia treatment further comprises administering other chemotherapy to the subject.

[0031] 21. The method of claim 20, wherein administering other chemotherapy to the subject comprises administering a chemotherapeutic agent selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof.

[0032] 22. The method of claim 21, wherein the chemotherapeutic agent is administered to the subject at least once prior to the administration of the anti-GPC3-CAR T cells.

[0033] 23. The method of claim 1, wherein the lymphopenia treatment reduces the number of lymphocytes by at least about 20%, the number of lymphocytes being measured by complete blood cell count (CBC) analysis.

[0034] 24. The method of claim 2, further comprising administering the anti-GPC3-CAR T cells to the subject for a second time.

[0035] 25. The method of claim 1, wherein the subject suffers from a refractory, persistent, or progressive disease.

[0036] 26. The method of claim 2, wherein the anti-GPC3-CAR T cells are autologous or allogeneic relative to the subject.

[0037] 27. The method of claim 1, further comprising administering at least one immunostimulant to the subject concurrently with or after the administration of the anti-GPC3 chimeric antigen receptor immune response cells.

[0038] 28. The method of claim 27, wherein the immunostimulant is selected from aldehyde interleukin (IL-2), IL-3, IL-6, IL-11, GM-CSF, and any combination thereof.

[0039] 29. The method of claim 19, wherein the biological agent is an antibody targeting an antigen expressed on lymphocytes.

[0040] 30. A kit for administering anti-GPC3-CAR immune-response cells to a subject exhibiting a solid tumor, comprising:

[0041] (a) an effective amount of anti-GPC3-CAR immune-response cells;

[0042] (b) a chemotherapeutic agent that effectively reduces the number of lymphocytes present in the subject; and

[0043] (c) instructions for administering the anti-GPC3-CAR immune-response cells to the subject after or simultaneously with the chemotherapeutic agent.

[0044] 31. The kit of claim 30, wherein the anti-GPC3-CAR immune-response cells are NK cells or T cells (anti-GPC3-CAR T cells).

[0045] 32. The kit of claim 30, wherein the chemotherapeutic agent is selected from cyclophosphamide, fludarabine, etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof.

[0046] 33. The kit of claim 30, further comprising about 60 mg / kg to about 80 mg / kg of cyclophosphamide or about 25 mg / m² to about 35 mg / m² of fludarabine formulated for administration to a subject in need.

[0047] 34. The kit of claim 31, comprising about 1 x 10⁸ to about 1 x 10¹¹ anti-GPC3-CAR T cells.

[0048] 35. The kit of claim 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells after administration of the chemotherapeutic agent.

[0049] 36. The kit of claim 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells at least 12 hours after administration of the chemotherapeutic agent.

[0050] 37. The kit of claim 31, wherein the instructions provide a procedure for administering the anti-GPC3-CAR T cells at least 24 hours after administration of the chemotherapeutic agent.

[0051] 38. The kit of claim 31, wherein the anti-GPC3-CAR T cells are formulated for intravenous injection.

[0052] 39. The kit of claim 31, wherein the anti-GPC3-CAR T cells are formulated for intra-arterial injection into the liver of the subject containing the solid tumor.

[0053] All publications, patents and patent applications mentioned herein are incorporated herein by reference to the extent specifically and individually.Each individual publication, patent, or patent application is incorporated herein by reference. In the event of any conflict between the terminology used herein and that used in the incorporated references, the terminology used herein shall prevail. Brief Description of the Drawings

[0054] The novel features of this disclosure are set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be gained by referring to the following detailed description and drawings illustrating illustrative embodiments in which the principles of this disclosure may be utilized; in the drawings:

[0055] Figure 1 shows a second-generation CAR-T vector encoding a chimeric antigen receptor targeting GPC3.

[0056] Figure 2 shows the ratio of lymphocytes to baseline levels in individuals HO1, HO2, or HO3 treated with cyclophosphamide and / or fludarabine.

[0057] Figure 3 shows magnetic resonance imaging (MRI) scans of liver sections before and after treatment.

[0058] Figure 4A shows the CytoTox non-radioactive cytotoxicity assay performed on 33-28BBZ, 92-28BBZ and 4-28BBZ co-cultured with control cells SK-HEP-1, and Figure 4B shows the CytoTox non-radioactive cytotoxicity assay performed on 33-28Z, 92-28Z, 92-BBZ and 92-28BBZ co-cultured with the second control cell line CHO-K1, with effector:target ratios of 3:1, 1:1 or 1:3.

[0059] Figure 5A shows the CytoTox non-radioactive cytotoxicity assay performed on 33-28BBZ, 92-28BBZ, and 4-28BBZ co-cultured with GPC3-positive hepatocellular carcinoma cells Huh-7, while Figure 5B shows the assay performed on 33-28BBZ, 92-28BBZ, and 4-28BBZ co-cultured with control cells transduced to express GPC3, CHO-K1-GPC3+, with effector:target ratios of 3:1, 1:1, or 1:3.

[0060] Figure 6A shows the CytoTox non-radioactive cytotoxicity assay performed on anti-GPC3 CAR-T cells (92-28Z, 92-BBZ, 92-28BBZ) or an empty vector co-cultured with HepG2 (HCC). Figure 6B shows the CytoTox non-radioactive cytotoxicity assay performed on anti-GPC3 CAR-T cells co-cultured with Hep3B (HCC) at 92-28Z, 92-BBZ, 92-28BBZ, or empty vector. Figure 6C shows the CytoTox non-radioactive cytotoxicity assay performed on anti-GPC3 CAR-T cells co-cultured with PLC / PRF / 5 (hepatocellular carcinoma) at 92-28Z, 92-BBZ, 92-28BBZ, or empty vector.

[0061] Figure 7A shows the CytoTox non-radioactive cytotoxicity assay performed on anti-GPC3 CAR-T cells co-cultured with Huh-7 cells.CytoTox non-radioactive cytotoxicity assays performed on CAR-T cells 33-28Z or 33-28BBZ (effectant:target ratio of 3:1, 1:1, or 1:3). Figure 7B shows CytoTox non-radioactive cytotoxicity assays performed on anti-GPC3 CAR-T cells 92-28Z or 92-28BBZ co-cultured with the gastric cancer cell line KATO-III (effectant:target ratio of 3:1, 1:1, or 1:3).

[0062] Figure 8A shows mice treated with second-generation CAR-T 92-28Z with significantly reduced tumor size compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8B shows tumor images showing mice transplanted with Huh-7 cells treated with second-generation CAR-T 92-28Z with significantly reduced tumor size compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8C shows that mice treated with second-generation CAR-T 92-28Z also had significantly reduced tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 8D shows that mice treated with second-generation CAR-T 92-28Z also had significantly reduced inhibition compared to mice treated with third-generation CAR-T 92-28BBZ or saline.

[0063] Figure 9 shows the specific dissolution results of various second- and third-generation anti-GPC3 CAR constructs 4-28Z, 4-28BBZ, 4-4-28BBZ, 4-14-28BBZ, 4-20-28BBZ, 4-35-28BBZ, and 4-42-28BBZ co-cultured with Huh7 (HCC) cells using a cytoTox non-radioactive cytotoxicity assay, with an effector:target ratio of 3:1 or 1:1.

[0064] Figure 10A shows the non-radioactive CytoTox cytotoxicity assay performed on anti-GPC3 CAR-T cells 33-28Z, 92-28Z, 92-BBZ, 92-28BBZ, or an empty vector co-cultured with A431 (GPC3 negative). Figure 10B shows the non-radioactive CytoTox cytotoxicity assay performed on anti-GPC3 CAR-T cells 33-28Z, 92-28Z, 92-BBZ, 92-28BBZ, or an empty vector co-cultured with Huh-7 (GPC3 positive). All CytoTox assays were performed at effector:target ratios of 3:1, 1:1, or 1:3.

[0065] Figure 11A shows the results of the xenograft mouse model, in which mice treated with second-generation CAR-T 92-28Z had a significantly reduced tumor volume (mm3) compared with mice treated with third-generation CAR-T 92-28BBZ or empty vector. Figure 11BThe results show that mice treated with second-generation CAR-T 92-28Z had significantly reduced tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline. Figure 11C shows tumor images from mice treated with empty vector, 92-28BBZ, or 92-28Z CAR-T. Detailed Description

[0066] The following description and examples detail embodiments of this disclosure. It should be understood that this disclosure is not limited to the specific embodiments described herein and therefore can be varied. Those skilled in the art will recognize that many variations and modifications are possible within the scope of this disclosure. Unless otherwise stated, any embodiment may be combined with any other embodiment.

[0067] As used herein, unless otherwise stated, some embodiments of the invention herein take into account numerical ranges. Various aspects of the invention may be presented in range format. It should be understood that the range format description is for convenience and brevity only and should not be construed as an inflexible limitation of the scope of the invention. Therefore, it should be considered that the description of the range has disclosed all possible subranges and individual numerical values ​​within that range as expressly written. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. When a range exists, the range includes the endpoints of the range. Specification 6 / 55 page 9 CN 121102467 A Definitions

[0068] As used herein, unless otherwise stated, the article “a” means one or more, unless otherwise expressly specified.

[0069] As used herein, unless otherwise stated, terms such as “comprising,” “containing,” “including,” etc., mean “including.”

[0070] The term “activation” and its grammatical equivalents as used herein can refer to the process by which a cell transitions from a quiescent state to an active state. This process may include a response to an antigen, migration, and / or phenotypic or genetic alteration to a functionally active state. For example, the term “activation” can refer to the stepwise process of T cell activation. For example, T cells may require at least two signals to become fully activated. The first signal may occur after the TCR is bound by the antigen-MHC complex, and the second signal may occur via the binding of co-stimulatory molecules (Table 3). In vitro, anti-CD3 can mimic the first signal and anti-CD28 can mimic the second signal. For example, engineered T cells can be activated by expressed CARs. As used herein, “T cell activation” or T cell triggering can refer to the state of T cells that have been adequately stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0071] As used herein, the term “antigen-binding unit” refers to immunoglobulin molecules and the immune binding of immunoglobulin molecules.An antigen-binding unit is an active part of an immunoglobulin (i.e., a molecule containing an antigen-binding site that specifically binds to an antigen (“immune response”). The term “antigen-binding unit” also includes immunoglobulin molecules from various species, including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibody (e.g., IgG) consists of four polypeptide chains: two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Immunoglobulins represent a large family of molecules, including several types such as IgD, IgG, IgA, IgM, and IgE. The term “immunoglobulin molecule” includes, for example, hybrid antibodies or modified antibodies and their fragments. It has been shown that the antigen-binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as “antigen-binding units.” The term “antigen-binding unit” also includes any molecular structure containing polypeptide chains with a specific shape suitable for recognizing epitopes, wherein one or more non-covalently binding interactions stabilize the complex between the molecular structure and the epitope.

[0072] If an antigen-binding unit binds to an antigen with greater affinity or affinity than other reference antigens, including peptides or other substances, then it “specifically binds” or “responds” to the antigen.

[0073] As used herein, “antigen” refers to a substance that is specifically recognized and bound by an antigen-binding unit. Antigens may include peptides, proteins, glycoproteins, polysaccharides, and lipids; portions thereof and combinations thereof. Non-limiting exemplary antigens include GPC3 and other homologs from humans and mice. “Antigen” may also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune competent cells or both. Those skilled in the art will understand that any macromolecule (including virtually all proteins or peptides) can act as an antigen.

[0074] As used herein, the term “immunoglobulin” or “Ig” can refer to a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as chimeric antigen receptors or antigen receptors. This class of proteins includes five members: IgA, IgG, IgM, IgD, and IgE, of which IgG is the most common circulating antibody. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody responses, and is crucial in the defense against bacteria and viruses. For example, CARs can recognize tumor cell antigens.

[0075] The term "anti-GPC3 antibody" can refer to an antibody or antibody-binding site that is capable of binding to GPC3 with sufficient affinity so that the antibody can be used to distinguish GPC3 from other antigens expressed by the cell. In one embodiment, the anti-GPC3 antibody binds to less than about 10% of the antibody binding to GPC3, for example, as measured by radioimmunoassay (RIA). In some embodiments, the antibody binding to GPC3 may have <1 μM, <100 nM, <10 nM, <5 nM,Dissociation constants (Kd) of <4 nM, <3 nM, <2 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.001 nM (e.g., 10⁻⁸ M or less, e.g., 10⁻⁸ M to 10⁻¹³ M, e.g., 10⁻⁹ M to 10⁻¹³ M). In some embodiments, the anti-GPC3 antibody binds to an epitope of GPC3 that is conserved among GPC3 from different species. Specification 7 / 55 pages 10 CN 121102467 A

[0076] The term “autologous” as used herein and its grammatical equivalents may refer to originating from the same organism. For example, a sample (e.g., cells) may be removed, processed, and returned to the same subject (e.g., patient) at a later time. Autologous processes are distinguished from allogeneic processes in which the donor and recipient are different subjects.

[0077] As used herein, “xenotransplantation” and its grammatical equivalents can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, wherein the recipient and the donor are different species. The transplantation of cells, organs, and / or tissues described herein can be used for xenotransplantation into humans. Xenotransplantation includes, but is not limited to, vascularized xenotransplantation, partially vascularized xenotransplantation, non-vascularized xenotransplantation, xenod dressings, xenobandages, and xenostructures.

[0078] As used herein, “allogeneic transplantation” and its grammatical equivalents (e.g., allogeneic transplantation) can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, wherein the recipient and the donor are different individuals of the same species. The transplantation of cells, organs, and / or tissues described herein can be used for allogeneic transplantation into humans. Allogeneic transplantation includes, but is not limited to, vascularized allogeneic transplantation, partially vascularized allogeneic transplantation, non-vascularized allogeneic transplantation, allogeneic dressings, allogeneic bandages, and allogeneic structures.

[0079] The term “autologous transplantation” as used herein and its grammatical equivalents (e.g., autologous transplantation) can include any procedure involving the transplantation, implantation, or infusion of cells, tissues, or organs into a recipient, wherein the recipient and the donor are the same individual. The transplantation of cells, organs, and / or tissues described herein can be used for autologous transplantation into humans. Autologous transplantation includes, but is not limited to, vascularized autologous transplantation, partially vascularized autologous transplantation, non-vascularized autologous transplantation, autologous dressings, autologous bandages, and autologous structures.

[0080] The term “chimeric antigen receptor” or “CAR” as used herein refers to an engineered molecule that can be expressed by immune cells, including but not limited to T cells. When expressed in T cells, CARs can redirect T cells to artificially...Receptor-directed specificity induces the killing of target cells. The extracellular binding domain of a CAR may be derived from mouse, humanized, or fully human monoclonal antibodies. “Anti-GPC3-CAR” is a CAR capable of binding to GPC3.

[0081] The term “epitope” and its grammatical equivalents as used herein may refer to an antigenic motif that can be recognized by an antibody, B cell, T cell, or engineered cell. For example, an epitope may be a cancer epitope recognized by a TCR. Multiple epitopes within an antigen may also be recognized. Epitopes may also be mutated.

[0082] The term “engineered” and its grammatical equivalents as used herein may refer to one or more alterations to nucleic acids (e.g., nucleic acids within the genome of an organism). The term “engineered” may refer to alterations, additions, and / or deletions of genes. Engineered cells may also refer to cells having added, deleted, and / or altered genes.

[0083] The term “cell” or “engineered cell” and its grammatical equivalents as used herein may refer to cells of human or non-human animal origin. Engineered cells may also refer to cells expressing CAR.

[0084] The term “Good Manufacturing Practice” (GMP) and its grammatical equivalents as used herein may refer to a product that is safe, effective, or pure according to FDA regulations. GMP may also be referred to as “cGMP”. “c” stands for “current”. Product manufacturers may use the latest technologies and systems to comply with GMP regulations for their products. GMP-compatible products are typically used in clinical settings, in contrast to research settings.

[0085] The term “transfection” as used herein refers to the introduction of foreign nucleic acids into eukaryotic cells. Transfection can be accomplished by a variety of means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipid transfection, protoplast fusion, retroviral infection, and biolistics.

[0086] The term “stable transfection” or “stable transfection” refers to the introduction and integration of foreign nucleic acids, DNA, or RNA into the genome of cells. The term "stable transfectant" refers to a cell in which foreign DNA has been stably integrated into the genomic DNA.

[0087] As used herein, the terms "encoding nucleic acid molecule," "encoding DNA sequence," and "encoding DNA" refer to the sequence or order of deoxyribonucleotides along a chain of deoxyribonucleic acid. The sequence of these deoxyribonucleotides determines the sequence of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0088] As used herein, the term "subject" refers to any animal, such as a mammal or marsupial. Subjects of the present invention include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any kind of poultry.

[0089] As used herein, the term “recipient” and its grammatical equivalents may refer to a person or non-human animal receiving a therapy or treatment.

[0090] As used herein, the term “peripheral blood lymphocyte” (PBL) and its grammatical equivalents may refer to lymphocytes circulating in the blood (e.g., peripheral blood). Peripheral blood lymphocytes may refer to lymphocytes not located in an organ. Peripheral blood lymphocytes may include T cells, NK cells, B cells, or any combination thereof.

[0091] The term “immune response cell” may refer to cells capable of initiating an immune response, including but not limited to T cells, B cells, and NK T cells, their respective precursor cells, and their progeny. Immune response cells may also refer to lymphoid or bone marrow lineage cells.

[0092] As used herein, the term “T cell” and its grammatical equivalents may refer to T cells of any origin. For example, T cells may be primary T cells, such as autologous T cells, cell lines, etc. T cells may also be human or non-human.

[0093] As used herein, the terms “T cell activation” or “T cell triggering” and their grammatical equivalents can refer to the state of T cells that have been adequately stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function. In some cases, “complete T cell activation” can be analogous to triggering T cell cytotoxicity. T cell activation can be measured using a variety of assays known in the art. Assays can be ELISA, ELISPOT for measuring cytokine secretion, flow cytometry for measuring intracellular cytokine expression (CD107), flow cytometry for measuring proliferation, and cytotoxicity assays (51Cr release assays) for determining target cell elimination. These assays typically use controls (unengineered cells) to compare with engineered cells (CAR T cells) to determine the relative activation of engineered cells compared to controls. Additionally, the assays can compare engineered cells incubated or in contact with target cells that do not express the target antigen. For example, CD19-CAR T cells incubated with target cells that do not express CD19 can be compared.

[0094] The term “sequence” and its grammatical equivalents, when used to refer to a nucleotide sequence, may include DNA or RNA; and may be single-stranded or double-stranded. Nucleic acid sequences may be mutated. Nucleic acid sequences may have any length, for example, from 2 to 1,000,000 or more nucleotides (or any integer value between or above), for example, from about 100 to about 10,000 nucleotides or from about 200 to about 500 nucleotides. Method of Use

[0095] In one aspect, methods for treating subjects exhibiting solid tumors expressing phosphatidylinositol proteoglycan-3 (GPC3) are disclosed herein. The subject method generally includes the step of administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or concurrently with subjecting the subject to lymphopenia treatment.

[0096] Subjects treated with the methods disclosed herein may exhibit cancer or solid tumors. Typically, cancer cells or solid tumor cells express one or more tumor antigens. Typically, the tumor antigen is phosphatidylinositol proteoglycan-3 (GPC3). Subjects exhibiting cancer or solid tumors expressing GPC3 may be referred to as having GPC3-positive cancer.

[0097] Cancers expressing GPC3 include, but are not limited to, liver cancer, gastric cancer, esophageal cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell cancer, melanoma, adenoma, adrenal cancer, schwannoma, malignant fibrous histiocytoma, or any combination thereof. The methods described herein are applicable to the treatment of squamous cell carcinoma or adenocarcinoma, gastrointestinal neuroendocrine carcinoma, or any other cancer disclosed in "Glypican-3 expression in gastrointestinal and pancreatic epithelial neoplasms. (2013) 44, 542-550 Human Pathology". Furthermore, the immune-response cells disclosed herein, such as the anti-GPC3-CAR-T cells, can be used to target ovarian cancer, cholangiocarcinoma, mesothelioma, breast cancer, lung squamous cell carcinoma, cervical intraepithelial neoplasia, cervical squamous cell carcinoma, intrahepatic and extrahepatic cancers, gallbladder cancer, invasive ductal carcinoma, clear cell carcinoma, large eosinophilic tumor, papillary carcinoma, adenocarcinoma, and lobular and medullary carcinoma of the breast. Other targets for anti-GPC3 therapy may include those found in “Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues. (2008) 129: 899-906 Am J Clin Pathol”.

[0098] In some cases, GPC3 expression in cancer cells or tumor cells can be assessed by flow cytometry or immunohistochemistry. GPC3 levels on cancer cells or tumor cells can be classified as low, intermediate, or high.

[0099] In some cases, cancer cells or tumor cells may express GPC3 on their cell surface. For example, GPC3 expression on the cell surface can be determined using antibodies against GPC3 using methods such as immunohistochemistry or flow cytometry analysis. Alternatively, GPC3 mRNA expression can be considered to be associated with GPC3 expression on the cell surface and can be determined by methods selected from in situ hybridization and RT-PCR.

[0100] In some cases, GPC3 is defined by a nucleus containing at least 50% sequence identity with the reference gene in Table 2.The gene encodes an acid sequence. GPC3 may be encoded by a gene containing a nucleic acid sequence that exhibits at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with the reference gene in Table 2.

[0101] In some embodiments, anti-GPC3 chimeric antigen receptor immune response cells are administered to a subject with a solid tumor expressing GPC3 after or simultaneously with lymphopenia treatment.

[0102] The subject anti-GPC3 chimeric antigen receptor (CAR) typically includes an extracellular antigen-binding region, a transmembrane domain, and an intracellular signaling domain that controls the activation of immune response cells. In some cases, the anti-GPC3 CAR further includes a hinge or spacer region. In some cases, the anti-GPC3 CAR further includes one or more co-stimulatory domains.

[0103] Chimeric antigen receptors typically include an extracellular antigen-binding region. In one embodiment, the extracellular antigen-binding region may be fully human. In other cases, the extracellular antigen-binding region may be humanized. In still other cases, the extracellular antigen-binding region may be mouse or chimeric, wherein the extracellular antigen-binding region consists of amino acid sequences from at least two different animal species. In some cases, the extracellular antigen-binding region may be non-human. Various antigen-binding regions can be engineered to target GPC3. Non-limiting examples include single-chain variable fragments (scFv) from antibodies, fragment antigen-binding regions (Fab) selected from libraries, single-domain fragments, or natural ligands that bind to their homologous receptors. Extracellular antigen-binding regions may include scFv, Fab, or natural ligands and any derivatives thereof. Extracellular antigen-binding regions may refer to molecules other than intact antibodies, which may include portions of intact antibodies and bind to antigens that can bind to intact antibodies. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0104] The extracellular antigen-binding region (e.g., scFv, Fab, or natural ligand) can be a portion of the CAR that determines antigen specificity. The extracellular antigen-binding region can bind to any complementary target. The extracellular antigen-binding region can be derived from an antibody with a known variable region sequence. The extracellular antigen-binding region can be derived from an antibody sequence obtained from a available mouse hybridoma. Alternatively, the extracellular antigen-binding region can be obtained from whole-exome sequencing of tumor cells or primary cells such as tumor-infiltrating lymphocytes (TILs). Specification 10 / 55 pages 13 CN 121102467 A

[0105] In some cases, the binding specificity of the extracellular antigen-binding region can be determined by the complementarity-determining region, or CDR (such as light...The binding specificity is determined by the light chain CDR or heavy chain CDR. In many cases, the binding specificity can be determined by the light chain CDR and the heavy chain CDR. A given combination of heavy chain CDR and light chain CDR can provide a given binding pocket that can confer greater affinity and / or specificity to an antigen such as GPC3 compared to other reference antigens. For example, a CDR specific to phosphatidylinositol proteoglycan-3 can be expressed in the extracellular binding region of a CAR so that a CAR targeting GPC3 can enable immune-responding cells to target tumor cells expressing GPC3.

[0106] In some aspects of any of the embodiments disclosed herein, the extracellular antigen-binding region such as scFv may contain a light chain CDR specific to GPC3. The light chain CDR may be a complementation-determining region of the light chain of an antigen-binding unit (such as the scFv of a CAR). The light chain CDR may comprise a sequence of consecutive amino acid residues, or a sequence of two or more consecutive amino acid residues separated by non-complementation-determining regions such as frame regions and optionally side-attached. In some cases, a light chain CDR may include two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. In some cases, a light chain CDR may include three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In some instances, a group of CDRs present on a common light chain may be collectively referred to as light chain CDRs.

[0107] In some aspects of any embodiment disclosed herein, an extracellular antigen-binding region such as scFv may include a heavy chain CDR that is specific for GPC3. The heavy chain CDR may be a complementation-determining region of the heavy chain of an antigen-binding unit (such as scFv). The heavy chain CDR may include a continuous sequence of amino acid residues, or two or more continuous sequences of amino acid residues separated by non-complementation-determining regions such as frames and optionally side-attached. In some cases, a heavy chain CDR may include two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. In some cases, a heavy chain CDR may include three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In some cases, a group of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.

[0108] In some cases, the extracellular antigen-binding region targeting GPC3 may be expressed by anti-GPC3 CAR immune response cells. In some cases, the CDR, light chain, and / or heavy chain binding to the GPC3 antigen may be contained within the extracellular antigen-binding region of CAR immune response cells such as CAR T cells. In some cases, the modified anti-GPC3 CDR may be expressed on the extracellular antigen-binding region of CAR immune response cells and have approximately 50% to approximately 100% homology with the original anti-GPC3 CDR. In some cases, the modified anti-GPC3 CDR may contain approximately 50%, 55%, 60%, 65%, or more of the unmodified anti-GPC3 CDR.70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% homology.

[0109] In some cases, GPC3-targeting scFvs may be expressed by anti-GPC3 CAR immune response cells. In some cases, CDRs, light chains, and / or heavy chains binding to the GPC3 antigen may be expressed on scFvs of CAR immune response cells (such as CAR T cells). In some cases, modified anti-GPC3 CDRs may be expressed on scFvs of CAR immune response cells such as CAR T cells and have about 50% to about 100% homology with the original anti-GPC3 CDR. In some cases, the modified anti-GPC3 CDR may contain about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% homology with the unmodified anti-GPC3 CDR. Table 1: Exemplary anti-GPC3 extracellular antigen binding regions: SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8

[0110] In a preferred embodiment, the extracellular antigen binding region specifically recognizes GPC3. GPC3 may contain a sequence that is at least 80% identical to the GPC3 gene referenced in Table 2. In some cases, the extracellular antigen binding region may target the N-terminus, C-terminus, or any portion from the N-terminus to the C-terminus of GPC3. The C-terminus of GPC3 may be covalently linked to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. In some cases, the C-terminus may contain about 1 to about 800 bases. The C-terminus may contain about 1, 50, 100, 150, 300, 500, 600, up to about 800 bases from the C-terminal end. In some cases, the extracellular antigen-binding region may target the GPI anchor of GPC3. Table 2: GPC3

[0111] The extracellular antigen-binding region can be modified in various ways by employing genetic engineering. In some cases, the extracellular antigen-binding region can be mutated to select an extracellular antigen-binding region with a higher affinity for its target. In some cases, the affinity of the extracellular antigen-binding region for its target can be optimized for a target that can be expressed at low levels in normal tissues. This optimization can be performed to minimize potential toxicity. In other cases, a clone of the extracellular antigen-binding region with a higher affinity for the membrane-bound form of the target may be superior to its soluble counterpart. This modification is possible because some targets can also be detected in soluble forms at varying levels, and their targeting can cause unintended toxicity.

[0112] In some cases, the extracellular antigen-binding region can be associated with SEQ ID NO.The extracellular antigen-binding region shares approximately 50% to approximately 100% homology with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some cases, the extracellular antigen-binding region may contain approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to approximately 100% homology with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some cases, the extracellular antigen-binding region may be mouse, humanized, or fully human. The extracellular antigen-binding region may be approximately 1% to approximately 100% human. In some cases, the extracellular antigen-binding region may be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or up to about 100% human.

[0113] In some cases, the extracellular antigen-binding region comprises a hinge or spacer region. The terms hinge and spacer region are used interchangeably. A hinge can be considered as a CAR portion used to provide flexibility for the extracellular antigen-binding region. In some cases, the hinge can be used to detect CARs on the cell surface of cells, particularly when antibodies to detect the extracellular antigen-binding region are ineffective or unavailable. For example, the length of the hinge derived from an immunoglobulin may need to be optimized depending on the location of the epitope on the target to which the extracellular antigen-binding region is targeted.

[0114] In some cases, the hinge may not belong to an immunoglobulin but to the natural hinge of another molecule, such as the CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues known to play a role in the interaction between the CD8 co-receptor and the MHC molecule. The cysteine ​​and proline residues can affect the performance of the CAR.

[0115] The CAR hinge can be size-adjustable and can be compensated to some extent to normalize the orthogonal synaptic distance between the CAR immune-responding cell and the target cell. This morphology of the immune synapse between the immune-responding cell and the target cell also defines the distance that cannot be functionally bridged by the CAR due to distal membrane epitopes on the cell surface target molecules, even with the use of short-hinge CARs, the synaptic distance cannot be brought close enough for signal transduction. Similarly, proximal membrane CAR target antigen epitopes have been described, where signal transduction output is observed only in the context of long-hinge CARs. The hinge can be adjusted according to the extracellular antigen-binding region used. The hinge can have any length.

[0116] Transmembrane domains can anchor the CAR to the cell's plasma membrane. The natural transmembrane portion of CD28 can be used for CARs. In other...In the case of CN 121102467 A, the native transmembrane portion of CD8α can also be used for CAR. “CD8” can mean a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with NCBI reference number NP_001759 or its stimulatory fragment. “CD8 nucleic acid molecule” can mean a polynucleotide encoding a CD8 polypeptide. In some cases, the transmembrane region can be the native transmembrane portion of CD28. “CD28” can mean a protein having at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with NCBI reference number NP_006130 or its stimulatory fragment. “CD28 nucleic acid molecule” can mean a polynucleotide encoding a CD28 polypeptide. In some cases, the transmembrane portion can include the CD8α region.

[0117] The intracellular signal transduction domain of a CAR can be responsible for activating at least one of the effector functions of an immune response cell in which the CAR has been placed. The CAR can induce effector functions of T cells, for example, cytotoxic activity or co-activation including cytokine secretion. Therefore, the term "intracellular signal transduction domain" refers to a protein portion that transduces effector function signals and directs the cell to perform specialized functions. While the entire intracellular signal transduction domain may generally be used, in many cases it is not necessary to use the entire chain of the signal transduction domain. In some cases, a truncated portion of the intracellular signal transduction domain is used. In some cases, the term "intracellular signal transduction domain" therefore means any truncated portion of the intracellular signal transduction domain that is sufficient to transduce effector function signals.

[0118] Preferred examples of signal transduction domains for CARs may include a T cell receptor (TCR) and a cytoplasmic sequence of a co-receptor that works together to initiate signal transduction upon target-receptor binding, as well as any derivatives or variants of these sequences, and any synthetic sequences having the same functional capabilities.

[0119] In some cases, the intracellular signaling domain may contain a signaling motif, referred to as an immune receptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. However, in a preferred embodiment, the intracellular signaling domain originates from the CD3ζ chain.

[0120] An example of a T cell signaling domain containing one or more ITAM motifs is the CD3ζ domain, also known as the T cell receptor T3ζ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays a crucial role in antigen recognition and...Several intracellular signal transduction pathways play an important role in coupling, which involves the activation of primary effectors of T cells. As used herein, CD3ζ primarily targets human CD3ζ and its isotypes (as known from Swissprot entry P20963), including proteins with substantially identical sequences. As part of a chimeric antigen receptor, the complete T cell receptor T3ζ chain is also not required, and any derivative containing the signal transduction domain of the T cell receptor T3ζ chain is suitable, including any of its functional equivalents.

[0121] The intracellular signal transduction domain may be selected from any of the domains in Table 3. In some cases, the domain may be modified such that the homology with any reference domain may be from about 50% to about 100%. Any of the domains in Table 3 may be modified such that the modified version may contain about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to about 100% homology.

[0122] The intracellular signaling domain of the CAR may further include one or more co-stimulatory domains. The intracellular signaling domain may include a single co-stimulatory domain, such as the ζ-chain (generation 1 CAR) or CD28 or 4-1BB (generation 2 CAR). In other instances, the intracellular signaling domain may include two co-stimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (generation 3).

[0123] Together with intracellular signaling domains such as CD8, these co-stimulatory domains can generate downstream activation of kinase pathways that support gene transcription and functional cellular responses. The co-stimulatory domains of the CAR can activate proximal signaling proteins associated with the CD28 (phosphatidylinositol-4,5-bisphosphate-3-kinase) or 4-1BB / OX40 (TNF-α receptor-associated factor adaptor) pathways, as well as MAPK and Akt activation.

[0124] In some cases, the signal generated by CAR can be combined with a secondary or co-stimulatory signal. Regarding the co-stimulatory domain, the chimeric antigen receptor-like complex can be designed to include several possible co-stimulatory domains. As is known in the art, in naive T cells, the involvement of T cell receptors alone is insufficient to induce complete activation of T cells into cytotoxic T cells. Complete and effective T cell activation requires a second co-stimulatory signal. Several receptors that have been reported to provide co-stimulation to T cell activation include, but are not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88, and 4-1BB. These co-stimulatory molecules utilize signal transduction pathways that share common characteristics with primary T cell receptor activation signals.These costimulatory signaling regions provide signals that can synergize with primary effector activation signals derived from one or more ITAM motifs (e.g., CD3ζ signaling domains) and fulfill the need for T cell activation.

[0125] In some cases, adding costimulatory domains to chimeric antigen receptor-like complexes can enhance the efficacy and durability of engineered cells. In another embodiment, T cell signaling domains and costimulatory domains are fused together to form a signaling region. Table 3. Costimulatory Domain Specification 14 / 55 pages 17 CN 121102467 A

[0126] In some cases, the subject CAR may contain a sequence or portion thereof that exhibits about 50% to about 100% sequence identity with any one of SEQ ID NO:9 to SEQ ID NO:13 in Table 4. In some cases, the subject CAR may contain a sequence that exhibits about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or up to about 100% sequence identity with any one of SEQ ID NO:9 to SEQ ID NO:13. Table 4: CAR-T domains containing exemplary hinges, transmembrane, and intracellular domains: SEQ ID NO: 9, 10, 11, 12, 13

[0127] In some cases, the subject CAR may contain a sequence that exhibits about 50% to about 100% sequence identity with any one of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 or SEQ ID NO: 30 (Table 5). In some cases, the subject CAR may contain SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:25.Any one of NO:30 that exhibits approximately 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or at most approximately 100% sequence identity. Table 5: Exemplary GPC3-resistant CAR SEQ ID NO Construct 14 4-28Z 15 4-28BBZ 16 4-4-28BBZ 17 4-4-28Z 18 4-14-28BBZ 19 4-14-28Z 20 4-20-28BBZ 21 4-20-28Z 22 4-35-28BBZ 23 4-35-28Z 24 4-42-28BBZ 25 4-42-28Z 26 33-28Z Specification 15 / 55 pages 18 CN 121102467 A 27 33-28BBZ 28 92-28Z 29 92-BBZ 30 92-28BBZ

[0128] In some cases, GPC3-resistant CARs may possess at least one or more of the following properties in any combination: a) binding to recombinant human GPC3; b) binding to recombinant cynomolgus monkey GPC3; c) binding to endogenous GPC3 on the surface of HepG2 cells; d) binding to cynomolgus monkey GPC3 expressed on the surface of 293 cells; e) binding to endogenous GPC3 on the surface of cancer cells; f) binding to endogenous GPC3 on the surface of hepatocellular carcinoma cells; g) binding to endogenous GPC3 on the surface of cell lines selected from HepG2, Hep3B, Huh7, and JHH-7; h) binding to epitopes within amino acids 25 to 137 of human GPC3; i) binding to trans-human GPC3. The epitope binding to the furin cleavage site at amino acid R358 / S359 of human GPC3; j) binding to the full-length mature human GPC3, but not to the N-terminal or C-terminal fragment of human GPC3; k) binding to the epitope within amino acids 420 to 470 of human GPC3; l) binding to the epitope within amino acids 470 to 509 of human GPC3; m) competing with antibody 7H1 for binding to human GPC3; n) competing with antibody 4G7 for binding to human GPC3; o) competing with antibody 15G1 for binding to human GPC3; binding to the C-terminal fragment of human GPC3; and / or p) competing with antibody 4A11 for binding to human GPC3.

[0129] The transgene encoding the subject anti-GPC3 CAR can be incorporated into cells. For example, the transgene can be incorporated into immune-responding cells such as T cells. When inserted into a cell, the transgene can be a complementary DNA (cDNA) segment, a copy of messenger RNA (mRNA), or the gene itself residing in its original genomic DNA region (with or without introns).

[0130] Nucleic acid (e.g., DNA) encoding a transgenic sequence can be randomly inserted into the chromosome of a cell. Random integration can be achieved by any method of introducing nucleic acid (e.g., DNA) into a cell. For example, such methods can be, but are not limited to, electroporation, acoustic pore effect, use of a gene gun, lipid transfection, calcium phosphate transfection, use of dendritic polymers, microinjection, and use of viral vectors including adenovirus, AAV, and retroviral vectors, and / or group II ribonucleases.

[0131] DNA encoding a transgenic sequence can be introduced into a cell via electroporation. DNA can also be introduced into a cell via lipid transfection, infection, or transformation. Electroporation and / or lipid transfection can be used to transfect primary cells. Electroporation and / or lipid transfection can be used to transfect primary hematopoietic cells. DNA can also be introduced into the cellular genome without the use of homologous recombination. In some cases, the flanking parts of the DNA can be engineered sites complementary to targeted double-strand break regions in the genome. In some cases, DNA can be excised from a polynucleotide such that it can be inserted into double-strand break regions without homologous recombination.

[0132] The transgene to be inserted may be flanked by engineered sites similar to targeted double-strand break sites in the genome to excise the transgene from the polynucleotide, thereby allowing it to be inserted into the double-strand break region.

[0133] The DNA encoding the transgene may also be designed to include a reporter gene, thereby allowing the presence of the transgene or its expression product to be detected by activating the reporter gene. Any reporter gene (such as those disclosed above) may be used. Cells containing the transgene may be selected by selecting cells in a cell culture in which the reporter gene has been activated.

[0134] CAR expression may be verified by expression assays (e.g., qPCR) or by measuring RNA levels. Expression levels may also indicate copy number. For example, if the expression level is very high, this may indicate that more than one copy of the CAR has been integrated into the genome. Alternatively, high expression may indicate that the transgene is integrated into a highly transcribed region, for example, near a highly expressed promoter. Expression may also be verified by measuring protein levels, such as by Western blotting.

[0135] Subjective anti-GPC3 CAR immune response cells may contain one or more transgenes. One or more transgenes may express a CAR protein that recognizes and binds to at least one epitope (e.g., GPC3) on an antigen, or binds to a mutated epitope on the antigen. The CAR may be a functional CAR. The subject anti-GPC3 CAR immune response cell may also contain one or more CARs, or it may contain a single CAR and a secondary engineered receptor. Specification 16 / 55 pages 19 CN 121102467 A

[0136] The transgene may encode a suicide gene. As demonstrated in many effective treatments in cancer patients, target tumor regression in response to CAR immune response cells may be accompanied by toxicity. In some cases, when the target antigen is present in the tumor and normal tissueWhen shared between tissues, CAR immune response cells may be unable to distinguish between tumor and normal tissue (“target-on-target / tumor-off-target” toxicity). In other cases, a systemic perturbation of the immune system, known as cytokine release syndrome (CRS), can occur. CRS can include systemic inflammatory response syndrome or cytokine storm, which may result from the rapid in vivo expansion of CAR immune response cells. CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure in severe cases. In most cases, the toxicity is associated with the in vivo expansion of infused CAR immune response cells, which can cause a general perturbation of the immune system and release high levels of pro-inflammatory cytokines such as TNFα and IL-6.

[0137] In some cases, CAR immune response cells targeting antigens shared with normal tissues can be generated, such that they transiently express CAR, for example, after electroporation of mRNA encoding the receptor. Furthermore, significant efforts have been made to further engineer CAR immune response cells by including a safety switch that allows for significant elimination of CAR immune response cells in cases of severe target-on-target toxicity. The vector encoding the CAR can be combined with a safety switch, such as an inducible cysteine-9 gene (activated by a dimerizing chemical inducer) or a truncated form of the EGF receptor R (activated by the monoclonal antibody cetuximab) or RQR8.

[0138] The subject anti-GPC3 CAR immune response cells may encode a suicide gene transgene. The transgene may also include a CAR receptor or another similar receptor. The suicide gene can induce the elimination of the CAR immune response cells. The suicide gene can be any gene that induces apoptosis in the CAR immune response cells. The suicide gene may be encoded within a viral vector together with the anti-GPC3 CAR.

[0139] One or more transgenes may be derived from different species. For example, one or more transgenes may include human genes, mouse genes, rat genes, pig genes, bovine genes, dog genes, cat genes, monkey genes, chimpanzee genes, or any combination thereof. For example, the transgene may be derived from humans and have a human gene sequence. One or more transgenes may include human genes. In some cases, one or more transgenes are not adenovirus genes.

[0140] As described above, transgenes can be inserted into the genome of immune-responding cells in a random or site-specific manner. For example, a transgene can be inserted into a random locus in the genome of an immune-responding cell. These transgenes can be functional, for example, fully functional if inserted at any location in the genome. For example, a transgene can encode its own promoter, or can be inserted at a location controlled by an endogenous promoter. Alternatively, a transgene can be inserted into a gene, such as an intron or exon, promoter, or non-coding region of a gene. The insertion of a transgene can cause the gene to be interrupted, for example, an endogenous immune checkpoint.

[0141] Sometimes, more than one copy of a transgene can be inserted into more than one random locus in the genome. For example, multiple copies can be inserted into random loci in the genome. This can lead to an increase in overall expression compared to a case where the transgene is randomly inserted once. Alternatively, one copy of the transgene can be inserted into a gene, and another copy of the transgene can be inserted into a different gene. The transgene can be targeted so that it can be inserted into a specific locus in the genome of an immune-responding cell.

[0142] In some cases, the polynucleotide containing the sequence encoding the subject anti-GPC3 CAR can be in the form of a plasmid vector. The plasmid vector may contain a promoter. In some cases, the promoter may be constitutive. In some cases, the promoter may be inducible. The promoter may be or may be derived from CMV, U6, MND, or EF1a. In some cases, the promoter may be adjacent to the CAR sequence. In some cases, the plasmid vector further contains a splice acceptor. In some cases, the splice acceptor may be adjacent to the CAR sequence. The promoter sequence may be a PKG or MND promoter. The MND promoter may be a synthetic promoter containing the U3 region of a modified MoMuLV LTR and a myeloproliferative sarcoma virus enhancer.

[0143] In some cases, the polynucleotide encoding the subject anti-GPC3 CAR may be designed for delivery to cells via non-viral techniques. In some cases, the polynucleotide may be a Good Manufacturing Practice (GMP) compatible reagent.

[0144] Expression of the polynucleotide encoding the subject anti-GPC3 CAR may be controlled by one or more promoters. The promoter may be ubiquitous, constitutive (unregulated promoters that allow the associated gene to be transcribed sequentially), tissue-specific, or inducible promoters. The expression of transgenes inserted near or around the promoter may be regulated. For example, the transgene may be inserted around or adjacent to a ubiquitous promoter. Some ubiquitous promoters may be the CAGGS promoter, hCMV promoter, PGK promoter, SV40 promoter, or ROSA26 promoter.

[0145] The promoter may be endogenous or exogenous. For example, one or more transgenes may be inserted near or around an endogenous or exogenous ROSA26 promoter. Furthermore, the promoter may be specific to immune-responding cells. For example, one or more transgenes may be inserted near or around a porcine ROSA26 promoter.

[0146] Tissue-specific or cell-specific promoters can be used to control the location of expression. For example, one or more transgenes may be inserted near or around a tissue-specific promoter. Tissue-specific promoters may be FABP promoters, Lck promoters, CamKII promoters, CD19 promoters, keratin promoters, albumin promoters, aP2 promoters, pancreatic islets, etc.Insulin promoter, MCK promoter, MyHC promoter, WAP promoter, or Col2A promoter.

[0147] Tissue-specific promoters or cell-specific promoters can be used to control the location of expression. For example, one or more transgenes can be inserted near or around a tissue-specific promoter. Tissue-specific promoters can be FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP promoter, or Col2A promoter.

[0148] Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing an inducer when needed. Inducible promoters can be, but are not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and / or Trex.

[0149] In addition, although not required for expression, the transgenic sequence may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and / or polyadenylation signals.

[0150] In some cases, the transgene encodes the subject anti-GPC3 CAR, wherein the transgene is inserted into a safe harbor, resulting in the expression of the anti-GPC3 CAR. In some cases, the transgene is inserted into the PD1 and / or CTLA-4 loci. In other cases, the transgene is delivered to cells in lentiviruses for random insertion, and PD1 or CTLA-4 specific nucleases may be provided as mRNA. In some cases, the transgene is delivered via viral vector systems such as retroviruses, AAVs, or adenoviruses along with mRNA encoding nucleases specific to the safe harbor (e.g., AAVS1, CCR5, albumin, or HPRT). Cells may also be treated with mRNA encoding PD1 and / or CTLA-4 specific nucleases. In some cases, the polynucleotide encoding the CAR is delivered via a viral delivery system along with mRNA encoding an HPRT-specific nuclease and a PD1 or CTLA-4-specific nuclease. CARs that can be used with the methods and compositions disclosed herein may include all types of these chimeric proteins, including first-, second-, and third-generation designs. Other agents such as CCR2 or siRNA may be applied to reduce PD-1 expression.

[0151] In some cases, a retroviral vector (γ-retrovirus or lentivirus) may be used to introduce the transgene into immune-responding cells. For example, a transgene encoding a CAR (e.g., an anti-GPC3 CAR) or any receptor or variant or fragment thereof that binds to the GPC3 antigen may be cloned into a retroviral vector and may be delivered by its endogenous promoter, a retroviral long terminus, or other means.Expression can be driven by end-repetitive or promoter-specific to the target cell type of interest. Non-viral vectors can also be used. Non-viral vector delivery systems may include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vectors such as liposomes or poloxamers.

[0152] Many virus-based systems have been developed to transfer genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged in retroviral particles using techniques known in the art. Vectors from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have an additional advantage over vectors from oncogenetic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells. They also have the additional advantage of low immunogenicity. Adenoviral vectors have the advantage of not integrating into the genome of the target cell, thereby avoiding negative integration-related events.

[0153] Cells can be transfected with a transgene encoding a CAR. The transgene concentration can be from about 100 picograms to about 50 micrograms. In some cases, the amount of nucleic acids (e.g., ssDNA, dsDNA, RNA) that can be introduced into the cells can be varied to optimize transfection efficiency and / or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample to power pores. In some cases, the amount of nucleic acids (e.g., dsDNA) required for optimal transfection efficiency and / or cell viability can be cell type specific. In some cases, the amount of nucleic acids (e.g., dsDNA) used for each sample can directly correspond to transfection efficiency and / or cell viability. For example, a range of transfection concentrations. The transgene encoded by the vector can be integrated into the cell genome. In some cases, the integration of the transgene encoded by the vector is positive. In other cases, the integration of the transgene encoded by the vector is negative.

[0154] In some cases, the starting cell density for viral delivery of cell modifications such as CAR can be varied to optimize transfection efficiency and / or cell viability. In some cases, the starting cell density for transfecting or transducing cells with a viral vector may be less than about 1 x 10⁵ cells. In some cases, the starting cell density for cell modification with a viral vector may be from at least about 1 x 10⁵ cells to at least about 5 x 10⁷ cells. In some cases, the starting cell density for optimal transfection efficiency and / or cell viability may be cell type specific. For example, for macrophages, a starting cell density of 1.5 x 10⁶ cells may be optimal (e.g., providing the highest viability and / or transfection efficiency). In another instance, for human cells, a starting cell density of 5 x 10⁶ cells may be optimal (e.g., providing the highest viability and / or transfection efficiency).In some cases, a certain range of starting cell densities may be optimal for a given cell type. For example, for human cells such as T cells, a starting cell density of 5.6 x 10⁶ to 5 x 10⁷ cells may be optimal (e.g., providing the highest viability and / or transfection efficiency).

[0155] The efficiency with which the nucleic acid sequence encoding the CAR is integrated into the cell genome using, for example, a viral system may be or may be about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher than 99.9%. In some cases, the CAR detection on the cell membrane of engineered cells can be or can be approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher than 99.9%, as measured by flow cytometry.

[0156] In some cases, the immune response cells may be stem memory TSCM cells composed of CD45RO(−), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+ and / or IL-7Rα+, which may also express CD95, IL-2Rβ, CXCR3 and / or LFA-1 and exhibit many functional properties different from those of the stem memory cells. Alternatively, the immune response cells may also be central memory TCM cells containing L-selectin and CCR7, wherein the central memory cells may secrete, for example, IL-2, but not IFNγ or IL-4. The immune response cells may also be effector memory TEM cells containing L-selectin or CCR7 and producing, for example, effector cytokines such as IFNγ and IL-4.

[0157] The carrier may be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or local administration, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells removed from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then the cells are typically re-implanted into the patient after selection of those incorporating the vector. Cells can be expanded before selection or after, as described on page 19 / 55 of the specification, 22 CN 121102467 A.

[0158] Suitable immune response cells for expressing anti-GPC3-CAR can be autologous or non-autologous cells for the recipient in need.

[0159] A suitable source of immune response cells can be obtained from the subject. In some cases, T cells can be obtained. The T cells can be obtained from many sources, including PBMCs, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, and tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of techniques known to those skilled in the art, such as Ficoll™, can be used to isolate T cells from blood units collected from the subject. In one embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes (including T cells, monocytes, granulocytes, and B cells), other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or culture medium for subsequent processing steps.

[0160] Alternatively, the cells can be from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In another embodiment, the cells can be part of a mixed population of cells exhibiting different phenotypic characteristics. Cell lines can also be obtained from transformed T cells according to the foregoing methods. Cells can also be obtained from cell therapy libraries. Modified cells resistant to immunosuppressive therapy can be obtained by any of the methods described herein. A desired cell population can also be selected prior to modification. An engineered cell population can also be selected after modification. Engineered cells can be used for autologous transplantation. Alternatively, cells can be used for allogeneic transplantation. In some cases, cells are administered to the same patient whose sample was used to identify cancer-related target sequences. In other cases, cells are administered to a different patient than the patient whose sample was used to identify cancer-related target sequences.

[0161] In some cases, the immune-responding cells can be primary cells, including primary T cells, stem cells, or progenitor cells. Progenitor cells can be hematopoietic progenitor cells. The cells of the present invention can be human cells. Suitable cells can be expanded in vitro. Suitable cells can also be CD45RO(−), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), IL-7Rα(+), or combinations thereof.

[0162] In some cases, suitable primary cells include peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subpopulations, such as, but not limited to, T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells, or non-pluripotent stem cells. In some cases, the cells can be any immune cell, including any T cell (TIL), such as tumor-infiltrating cells, such as CD3+ T cells, CD4+ T cells, CD8+ T cells, or any other type of T cell. T cells may also include memory T cells, memory stem T cells, or effector T cells. T cells may also be optional.T cells can be selected from a bulk population, for example, from whole blood. T cells can also be expanded from a bulk population. T cells can also be biased towards specific populations and phenotypes. For example, T cells may be phenotyped as containing CD45RO(−), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+). Suitable cells can be selected that contain one or more markers selected from the following: CD45RO(−), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and / or IL-7Rα(+). Suitable cells also include stem cells, for example, such as embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells. Suitable cells can include any number of primary cells, such as human cells, non-human cells, and / or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from the subject to be treated (e.g., a patient). Suitable cells may be derived from human donors. Suitable cells may be stem memory TSCM cells composed of CD45RO(−), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, which may also express CD95, IL-2Rβ, CXCR3, and LFA-1 and exhibit many functional properties different from those of the stem memory cells. Suitable cells may be central memory TCM cells containing L-selectin and CCR7, which may secrete, for example, IL-2 but not IFNγ or IL-4. Suitable cells may also be effector memory TEM cells containing L-selectin or CCR7 and producing, for example, effector cytokines such as IFNγ and IL-4.

[0163] In some cases, the method may include enriching cultured T cells with CD8+ T cells prior to rapid cell expansion. After culturing T cells in IL-2, CD8+ cells can be consumed and enriched using methods such as CD8 microbead separation (e.g., using the CliniMACSplus CD8 microbead system (Miltenyi Biotec)). Without being bound by any particular theory, it can be argued that CD4+, CD25+ regulatory T cells can impair antitumor responses. Therefore, enriching cultured T cells with CD8+ T cells and reducing or eliminating CD4+ cells can be considered to improve the effects of adoptive transfer of antitumor CD4+ cells, improve response rates in patients, and / or reduce toxicity seen through CD4+ cytokine production. Furthermore, enriched CD8+ “young” T cells can be considered...In rapid clinical-scale expansion, it performs more reliably and predictably than mixed T cells.

[0164] The cells may be Good Manufacturing Practice (GMP) compatible reagents. The cells may be part of a combination therapy to treat cancer, infection, autoimmune disease, or graft-versus-host disease (GVHD) in subjects in need. In some cases, the cells of the present invention may be administered as a monotherapy to subjects in need.

[0165] In some cases, the cells expressing the subject CAR comprise a heterogeneous population of T cells. In some cases, the cells used may consist primarily of CD4 and CD8 T cells in varying proportions. The CD4 and CD8 cells may have phenotypic characteristics of circulating effector T cells. The CD4 and CD8 cells may also have phenotypic characteristics of effector-memory cells. In another embodiment, the cells may be central-memory cells.

[0166] Suitable cells that can be isolated from a donor may be at any developmental stage, including but not limited to fetuses, newborns, young adults, and adults. For example, donor immune response cells may be isolated from adults. Donor immune response cells can be from individuals aged 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year. For example, immune response cells can be isolated from individuals under 6 years of age. Immune response cells can also be isolated from individuals under 3 years of age. Donors can be over 10 years of age.

[0167] Methods for obtaining suitable cells may include selection based on given markers. For example, such markers may include GFP, resistance genes, cell surface markers, or endogenous tags. Cells can be selected using any endogenous marker. Applicable cell selection techniques include flow cytometry and / or magnetic column chromatography. The selected cells can then be infused into the subject. The selected cells may also be expanded to a large number. The selected cells may be expanded prior to infusion.

[0168] The amount of cells necessary for therapeutic efficacy in a patient can vary depending on cell viability and the efficiency of cell genetic modification (e.g., the efficiency of transgene integration into one or more cells, or the expression level of proteins encoded by the transgene). In some cases, the products of genetically modified cell viability (e.g., proliferation) and the efficiency of transgene integration may correspond to a therapeutic aliquot of cells available for administration to a subject. In some cases, an increase in genetically modified cell viability may correspond to a reduction in the number of cells necessary for therapeutically effective administration in a patient. In some cases, an increase in the efficiency of transgene integration into one or more cells may correspond to a reduction in the number of cells necessary for therapeutically effective administration in a patient. In some cases, determining the number of cells necessary for therapeutic effectiveness may include determining a function corresponding to cell viability over time. In some cases, determining the number of cells necessary for therapeutic effectiveness may include determining a function corresponding to the ability of a transgene to integrate into a cell relative to time-related variables (e.g., cell culture time, electroporation time, cell stimulation time).The efficacy is a function of the variation in one or more cells. In some cases, therapeutically effective cells may be a cell population containing about 30% to about 100% anti-GPC3 CAR expression on the cell surface. In some cases, therapeutically effective cells may express about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% to more than about 99.9% anti-GPC3 CAR on the cell surface, as measured by flow cytometry.

[0169] As described above, various cells can be used to express the subject CAR. Anti-GPC3 CARs can be present in the plasma membrane of eukaryotic cells, such as mammalian cells, including but not limited to cytotoxic cells, T lymphocytes, stem cells, stem cell progeny, progeny cells, and NK cells.

[0170] When present in the plasma membrane of eukaryotic cells, the CAR can be active in the presence of its binding target. For example, an anti-GPC3 CAR can be active in the presence of GPC3. Targets, such as GPC3, can be expressed on the membrane. Targets can also be soluble (e.g., not cell-bound). Targets can be present on the surface of cells, such as target cells. Targets can be present on solid surfaces, such as lipid bilayers; etc. Targets can be soluble, such as soluble antigens. Targets can be antigens. Antigens can be present on the surface of cells, such as target cells. Antigens can be present on solid surfaces, such as lipid bilayers; etc. In some cases, the target can be an epitope of the antigen. In the methods disclosed herein, the antigen is typically GPC3, and the target cells expressing GPC3 are cancer cells or tumor cells.

[0171] In some cases, when the CAR is present on the cell's plasma membrane and when activated by binding its target, it can result in cytotoxic activity of the cell against the target, which expresses an antigen bound to the CAR-binding domain on its cell surface. For example, in some cases, the cell may be a cytotoxic cell (e.g., NK cells or cytotoxic T lymphocytes), and the CAR of this disclosure, when present on the cell's plasma membrane and when activated by binding its target, can increase the cytotoxic activity of the cytotoxic cell against the target cell, which expresses an antigen bound to the CAR-binding domain on its cell surface. For example, in some cases, the cell may be an NK cell or T lymphocyte, and the CAR of this disclosure, when present on the cell's plasma membrane and when activated by binding its target, can increase the cytotoxic activity of the cell by at least 10%, at least 15%, at least 20%, at least 25%, or at least [missing information] compared to the cytotoxic activity of the cell when the target is not bound.30%, at least 40%, at least 50%, at least 75%, at least 2 times, at least 2.5 times, at least 5 times, at least 10 times, or more than 10 times.

[0172] In some cases, when activated by binding to its target, CAR can lead to other CAR activation-related events, such as proliferation and expansion (due to increased cell division or anti-apoptotic responses). Cell proliferation can be visually measured by observing cell clusters seen under a microscope. Cell expansion can be measured by a hematology counter. In some cases, CAR-T cells may have increased cell proliferation and expansion compared to equivalent T cells (non-CAR T cells). The increased cell expansion can be from about 1 to about 20 times compared to equivalent cells. The increased cell expansion compared to equivalent cells can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 times. Cell expansion can be measured over a period of time. For example, cell expansion can be performed between cell collection and infusion into the subject. In other cases, cell expansion can be performed from 1 day to up to approximately 30 days after collection. Cell expansion can be performed approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or up to 30 days after collection. In some cases, cells can be expanded using a rapid expansion protocol (REP) prior to infusion into the subject. In some cases, REP can occur over a period of approximately 14 days.

[0173] In some cases, when activated by binding to its target, CAR can lead to other CAR activation-related events, such as regulation of intracellular signaling, cell differentiation, or cell death. In some cases, CAR expression on cells can alter intracellular signaling. In other cases, CAR expression can alter cell differentiation.

[0174] Subjective anti-GPC3 CAR immune response cells can be administered to cancer subjects or tumor subjects expressing GPC3 concurrently with or after lymphopenia treatment.

[0175] In some aspects, anti-GPC3 CAR immune response cells are administered after lymphopenia treatment. This treatment can reduce circulating lymphocytes in the treated subject, or substantially deplete circulating lymphocytes (i.e., lymphopenia). For example, anti-GPC3 CAR immune response cells can be administered from at least 1 hour to at least 1 week after lymphopenia treatment. For example, treatment can be administered at least 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, or 7 days or more after lymphopenia treatment.Prolonged administration of anti-GPC3 CAR immune response cells. In some cases, CAR-T may be administered approximately 1, 2, 3, 4, 5, 6 weeks or longer after lymphopenia treatment.

[0176] In some cases, host lymphopenia can promote the expansion of anti-GPC3 CAR immune response cells such as CAR T cells. On the one hand, lymphopenia can create “space” for upcoming adoptive transfer cells, and on the other hand, it can induce homeostatic expansion of these cells. The latter effect may be mediated by chemoablation of endogenous regulatory T cells, which typically secrete inhibitory cytokines (e.g., TGF-β and IL-10) that can limit the expansion of effector cells such as CAR T cells. In some cases, lymphopenia treatment can increase the in vivo expansion of anti-GPC3 CAR immune response cells by approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 times compared to treatment with anti-GPC3 CAR immune response cells without lymphopenia treatment.

[0177] Additionally, due to less competition and increased production of lymphocytes, T cell growth homeostatic cytokines such as IL-7 and IL-15, which are typically present in limited quantities, become readily available. Therefore, lymphopenia can be induced prior to infusion of anti-GPC3 CAR immune response cells to increase the efficacy of anti-GPC3 CAR immune response cells in treated subjects. In some cases, lymphopenia treatment measured by tumor reduction or control can increase antitumor efficacy by approximately 10% to approximately 100% compared to treatment without lymphopenia treatment. For example, compared to treatment without lymphopenia, lymphopenia treatment can increase antitumor efficacy by approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100%.

[0178] Lymphopenia can be performed using various methods. Total body irradiation (TBI) or cytotoxic drugs can be used to perform lymphopenia prior to the administration of anti-GPC3 CAR immune response cells. While these methods may be intended to deplete the recipient's lymphatic compartments, they can also promote the presentation of tumor antigens by triggering tumor cell death and antigen release. These antigens can then be accepted and presented by antigen-presenting cells (APCs) to enhance the activation of anti-GPC3 CAR immune response cells.

[0179] Radiation and / or chemotherapy can induce host cell activation, leading to the release of pro-inflammatory cytokines such as TNF-α, IL-1, and IL-4.Release and upregulation of co-stimulatory molecules such as CD80. In some cases, lymphopenic therapy can improve anti-GPC3 CAR immune response cell therapy by activating host cells. In some cases, lymphopenic therapy can improve anti-GPC3 CAR immune response cell therapy by upregulating co-stimulatory molecules. In addition to enhanced APC function and availability, premodulation protocols can also disrupt the integrity of the mucosal barrier by radiation-induced apoptosis of these organ lining cells. Damage to the gut can allow bacterial products such as LPS to translocate into the systemic circulation. LPS can in turn activate anti-GPC3 CAR immune response cells in vivo and can enhance anti-tumor responses. Thus, pro-inflammatory cytokines and microbial products provide key “danger signals” for the activation and maturation of DCs, thereby enhancing anti-GPC3 CAR immune response cell-mediated tumor therapy.

[0180] In some cases, lymphopenic therapy can selectively deplete cells expressing CD25 in the human body, including humanized anti-Tac (anti-CD25) and ONTAKTM (IL-2 conjugated with diphtheria toxin).

[0181] In some cases, chemotherapeutic agents may be administered to achieve lymphopenia in the subject. In some cases, the subject may receive nonmyeloablative lymphopenic chemotherapy. Nonmyeloablative lymphopenic chemotherapy may be any suitable such therapy, which may be administered via any suitable route. Chemotherapy regimens may include the use of a single alkylating agent, such as cyclophosphamide or chlorambucil, or combinations such as CVP (cyclophosphamide, vincristine, and prednisone), CHOP (CVP and doxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone, and procarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD (CHOP plus methotrexate, bleomycin, and leucovorin), ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide, and leucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone, doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, and leucovorin), and MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, fixed-dose prednisone, bleomycin, and leucovorin). Instructions for Use, Pages 23 / 55, CN 121102467 A

[0182] Non-myeloablative lymphopenic chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly if the cancer is GPC3 positive, it may be metastatic. The preferred route of administration of cyclophosphamide and fludarabine may be intravenous. Similarly, any suitable dose of cyclophosphamide and fludarabine may be administered. Preferably, about 60 mg / kg of cyclophosphamide may be administered for two days, followed by about 25 mg / m² of fludarabine for about five days.

[0183] In some cases, lymphopenia may be performed to minimize cellular rejection in the immune-mediated anti-GPC3CAR immune response. For example, a subject may be treated with cyclophosphamide (Cy) followed by fludarabine (Flu) lymphopenia treatment. The dosage of chemotherapeutic agents such as Cy can be from about 1 mg / kg to about 200 mg / kg. The dose of Cy in the subjects can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or up to approximately 200 mg / kg.

[0184] Alternatively, an exemplary dose and regimen for Cy treatment may be about 0.5–5 g / m² / day, preferably 0.6–3 g / m² / day, more preferably 1–2 g / m² / day; for 1–3 days, preferably 1–2 days;

[0185] The dose of Flu in the subject may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or up to approximately 200 mg / m2.

[0186] Alternatively, an exemplary dose and regimen for fludarabine treatment may be about 20–80 mg / m² / day, preferably 25–70 mg / m² / day or 25–30 mg / m² / day, for 2–10 days, 3–8 days, or 4, 5, 6, or 7 days.

[0187] In some cases, Cy and Flu may be used in combination. For example, the initial dose of Flu may be in the range of about 10–60 mg / m² / day, 15–50 mg / m² / day, or 20–30 mg / m² / day for 2–8 days or 3–6 days, followed by Cy administration at a dose of about 0.2–1 mg / m² / day, 0.3–0.8 mg / m² / day, or 0.4–0.6 g / m² / day for 1–5 days or 2–3 days.

[0188] In some cases, prior to infusion of anti-GPC3 CAR immune response cells, subjects may receive Cy and Flu treatment, with 60 mg / kg Cy administered once and 25 mg / m² Flu administered 3 to 5 times. Prior to infusion of anti-GPC3 CAR immune response cells, subjects may receive about 0 to about 20 doses of lymphodepletant. Prior to anti-GPC3 CAR immune response cell infusion, subjects may receive approximately 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to 20 doses of a lymphocyte-depleting agent such as Cy or Flu. In other cases, subjects may receive a lymphocyte-depleting agent, such as Cy and / or Flu, concurrently with anti-GPC3 CAR immune response cell infusion. In other cases, a lymphocyte-depleting agent such as Cy and / or Flu may be administered to subjects after anti-GPC3 CAR immune response cell infusion. In other cases, a lymphocyte-depleting agent may be administered to subjects before, concurrently with, and / or after anti-GPC3 CAR immune response cell infusion. In some cases, subjects may not receive lymphopenic therapy. In some cases, different doses of lymphocyte-depleting agents may be used during the course of the regimen. For example, a subject may receive 60 mg / kg Cy before receiving anti-GPC3 CAR immune response cells, and then receive 40 mg / kg Cy concurrently with receiving anti-GPC3 CAR immune response cells. A complete blood count (CBC) may be performed to determine the extent of lymphopenia and whether additional administration may be necessary. In some cases, Cy may be administered alone. In other cases, Flu may be administered alone. In some cases, Cy and Flu may be used alternately during the protocol.

[0189] Lymphopenia may improve the expansion of anti-GPC3 CAR immune response cells. Lymphopenia may improve the persistence of anti-GPC3 CAR immune response cells in the blood. The expansion and persistence of anti-GPC3 CAR immune response cells can be detected by flow cytometry analysis using anti-CAR antibodies. The persistence of anti-GPC3 CAR immune response cells can also be measured by evaluating the copy number of anti-GPC3 CAR immune response cells using qPCR.

[0190] In some cases, the acceptable dose of anti-GPC3 CAR immune response cells may be reduced if lymphopenia treatment is administered compared to subjects who may not have received lymphopenia treatment.

[0191] In some cases, lymphopenia treatment may be administered over a period of time. For example, lymphopenia treatment may be administered over a course of treatment from 1 minute to approximately 24 hours. Lymphopenia treatment may be administered for 1 minute, 15 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or up to approximately 24 hours. Lymphopenia agents may be administered in combination with other agents such as diluents, uroprotectants, excipients, or combinations thereof. For example, sodium 2-mercaptoethanesulfonate (mesna) may be included in lymphopenia treatment. In some cases, subjects may be given cyclophosphamide intravenously at a dose of 60 mg / kg / day in 250 ml D5W for approximately 2 days, or mesna at a dose of 15 mg / kg / day over 1 hour for 2 days. Mesna can be co-administered with various doses of lymphocyte-depleting agents. For example, mesna can be administered at doses from approximately 1 mg / kg / day up to approximately 50 mg / kg / day. Mesna can be administered at doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or up to approximately 50 mg / kg / day. In some cases, for obese or pediatric subjects, the dosage can be calculated using actual weight. Actual weight can be the average of actual weight and ideal weight. For example, for men, ideal weight can be calculated as = 50 kg + 2.3 (the number of inches over 60 inches). For women, ideal weight can be calculated as = 45.5 kg + 2.3 (the number of inches over 60 inches). In some cases, Flu administration may be 25 mg / m² / day via intravenous infusion (IVPB) over 30 minutes for 5 days. In some cases, Flu administration may be performed before Cy administration. In some cases, Flu administration may be performed concurrently with Cy administration. In some cases, Flu administration may be performed after Cy administration. In some cases, Flu darabine may be administered approximately 1 to 2 hours after Cy and mesna.

[0192] Whole-body radiation may take the form of radiotherapy. As the name suggests, TBI can involve radiation to the entire body, but in modern practice...In practice, the lungs may be partially shielded to reduce the risk of radiation-induced lung injury. Whole-body radiation can be administered in various doses. For example, whole-body radiation can be administered at a dose of about 10 to about 12 Gy. In some cases, whole-body radiation may be administered in fractions, delivering smaller doses in several smaller doses rather than delivering the entire dose at once. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 different doses of radiation may be administered.

[0193] The reported D0 value (the amount of ionizing radiation necessary to eradicate a specific cell type) for hematopoietic stem cells may be about 0.5 to about 1.4 Gy, while the D0 value for human leukemia cell lines may be about 0.8 to about 1.5 Gy, indicating that both cell types are radiosensitive. The ideal dosing regimen may depend on the patient's age, disease, and intended type of treatment. Myeloablative TBI may be administered at a dose of about 12 to about 15 Gy, in fractions of about 8 to about 12 over about 4 days, about 2 to 3 times daily. In some cases, myeloablative TBI may be about 5 to about 20 Gy. Myeloablative TBI can be administered in doses of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20 Gy. In some cases, myeloablative TBI can be administered in doses of about 5 to about 20. Myeloablative TBI can be administered in doses of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 to about 20. Myeloablative therapy can be administered over a period of about 1 to about 10 days. Myeloablative therapy can be administered over a period of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to about 10 days.

[0194] In some cases, low-dose TBI may comprise a dose of about 2 to about 8 Gy administered in doses of about 1 to about 4. In some cases, low doses may include about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or up to 8 Gy. Low-dose TBI may be administered to subjects who cannot tolerate bone marrow ablation due to age or comorbidities.

[0195] TBI may be administered using parallel opposing pairs of high-energy photons, the photon beam being about 4 to about 18 MV for TBI. In some cases, for TBI, the photon beam may be about 1 to about 25 MV. The photon beam may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or up to about 25 MV. TCI can be performed using devices such as the Varian Clinac iX or Siemens Artiste.

[0196] In some cases, the application rate can be from about 5 cGy / min to about 100 cGy / min. The application rate can be about5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 95 or up to 100 cGy / min.

[0197] In some cases, certain organs may be shielded during radiation. For example, the liver, lungs, brain, heart, or combinations thereof may be shielded during whole-body radiation. Biological agents

[0198] In some cases, biological agents such as antibodies may be used to deplete immune cells. Monoclonal antibodies (mAbs) that are lysogenic to lymphocytes may be another means of producing lymphopenia. In some cases, antibodies for T-cell lymphopenia that may be administered prior to infusion of anti-GPC3 CAR immune response cells should be effective but transient in vivo to allow for rapid infusion and proliferation of infused anti-GPC3 CAR immune response cells. The antibodies used may target markers expressed on the surface of cells such as lymphocytes. In some cases, lymphopenic antibodies may be anti-CD3, anti-CD4, anti-CD8, anti-CD45, anti-CD25, anti-CD52, or any combination thereof.

[0199] Antibodies such as alenzusmab (Campath-1H), bortezomib, thymoglobulin (rabbit ATG, Genzyme), ATGAM (horse ATG, Pfizer), and alenzusmab (Campath-1H) can be used. Rituximab (IDEC-C2B8), GA101, humanized anti-CD20 IgG1, XmAb5574, Fc-engineered anti-CD19 antibody, afasicept (LFA3-Ig), fingolimod (FTY720), anti-thymocyte globulin (ATG), anti-CD4 antibody, anti-CD3 antibody, and anti-CD8 antibody can also be used as lymphopenic agents. In some cases, cladribine (2-CdA), a purine analogue similar to fludarabine, can be used. In some cases, antibody lymphopenic therapy may comprise a single antibody (such as alenzusmab (anti-CD52)). In other cases, antibody-based lymphopenia treatment may include at least two antibodies, such as alenzumab and anti-CD45. In some cases, lymphopenia treatment may include multiple lymphopenic pathways, such as combination therapy with anti-CD45.Radiotherapy or chemotherapy combined with radiotherapy. Chemotherapy and antibody therapy may also be used in combination.

[0200] Antibiotics, antifungal agents and antiviral agents may be given to the subject as prophylaxis during lymphopenia treatment. For example, prophylaxis may include 500 mg Altrex daily, q MWF one tablet of cyproheptadine DS and 200 mg fluconazole daily. Drug treatment continues until the absolute lymphocyte count (ALC) and absolute neutrophil count (ANC) return to the baseline before drug treatment. Biological agents may also be doxorubicin.

[0201] One or more cytokines may be introduced with the cells. Cytokines may be used to promote the expansion of metastatic cells (including adoptive tumor-specific cells) within the tumor microenvironment. In some cases, IL-2 may be used to promote the expansion of the cells described herein. Cytokines such as IL-1 may also be used. Other relevant cytokines in the field of immunotherapy, such as IL-2, IL-7, IL-12 and L-21 or any combination thereof, may also be used. In some cases, recombinant cytokines are used.

[0202] In some cases, T-cell growth factors may be administered. Growth factors may be administered via any suitable route. If more than one T-cell growth factor is administered, they may be administered simultaneously or sequentially in any order, and via the same or different routes. T-cell growth factors such as interleukin-2 (IL-2) may be administered as an intravenous bolus. The dose of T-cell growth factors such as IL-2 is considered high by those skilled in the art. Preferably, a dose of about 720,000 IU / kg of IL-2 may be administered three times daily until tolerated. In some cases, about 5 to about 15 doses of IL-2 may be administered, with an average of about 9 doses. The dose of T-cell growth factors may be about 0 to about 20. T-cell growth factors may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about 20 times.

[0203] IL-2 can be administered as an intravenous bolus at a dose of 720,000 IU / kg (based on total weight). In some cases, IL-2 can be administered over 15 minutes. In some cases, IL-2 can be administered over 20 minutes. IL-2 can be administered by immediate injection. In some cases, IL-2 can be administered at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, or 29 minutes.The time intervals are 1 hour, 2 hours, 3 hours, 3 hours, 4 hours, 5 hours, or up to 6 hours.

[0204] In some cases, IL-2 may be initiated within 24 hours of cell infusion and continued for up to about 4 days (up to 12 doses). In some cases, IL-2 may be administered up to approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after initial administration.

[0205] The dose of IL-2 may be administered every 8 hours. In some cases, IL-2 may be administered approximately every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after initial administration. In some cases, IL-2 administration may be discontinued if toxicity is detected. In some cases, if a patient experiences grade 3 or 4 toxicity from adeleukin, the dose may be delayed or discontinued, unless it is a common reversible grade 3 toxicity of adeleukin, such as diarrhea, nausea, vomiting, hypotension, skin changes, anorexia, mucositis, dysphagia, or systemic symptoms and laboratory changes. In some cases, if these toxicities can be easily reversed within 24 hours with supportive measures, an additional dose may be given. Additionally, the attending physician may decide whether to maintain or discontinue administration.

[0206] In some cases, immune response cells expressing anti-GPC3 CAR may have increased antitumor efficacy compared to equivalent immune response cells that do not express anti-GPC3 CAR.

[0207] In some cases, antitumor efficacy may refer to cytotoxic activity. In other cases, antitumor efficacy may refer to persistence. Antitumor efficacy may also refer to the ability of cells to target tumors. Various in vitro assays can be used to measure antitumor efficacy. For example, cytotoxic capacity can be measured by an ELISA measuring the release of interleukin-2 (IL-2) or interferon-γ (IFNγ). Cytotoxic activity can be measured by killing assays such as chromium-51 release assays or co-culture assays. In some cases, anti-GPC3 CAR immune-responding cells may exhibit increased antitumor efficacy and capacity compared to comparable cells.

[0208] Anti-GPC3 can be evaluated using various methods.CAR therapeutic efficacy. Efficacy can refer to the degree of anti-tumor efficacy in controlling, reducing, or eliminating tumors, such as GPC3-positive tumors. Therapeutic efficacy can also refer to CAR immune response cell expansion, persistence, tumor targeting, and any combination thereof.

[0209] Subjects eligible for anti-GPC3 CAR immune response cell therapy, such as anti-GPC3 CAR T-cell therapy, can be evaluated during infusion, immediately after infusion, or years after infusion. For example, treated subjects can return to the clinic for evaluation from approximately one day to a lifetime. Subjects treated may be evaluated at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, or up to 90 years after initial administration of the subject-specific CAR immune response cells. In some cases, the evaluation plan may include daily, weekly, monthly, or annual monitoring. In some cases, subjects may be observed more frequently as clinically indicated. Evaluation may include physical examination, chemical evaluation, complete blood count, thyroid group, toxicity assessment, computed tomography (CT) scans of body regions, apheresis, and any combination thereof.

[0210] In some cases, apheresis may be performed approximately 1 to 10 weeks before and after administration of the subject CAR immune response cell infusion. At other time points, peripheral blood lymphocytes (PBLs) of the subject may be obtained from whole blood using purification by Ficoll gradient centrifugation. Aliquots of peripheral blood mononuclear cells (PBMCs) may be cryopreserved for immune monitoring of cell function. In some cases, various tests may be used to evaluate the immune relevance of the subject CAR immune response cell therapy, including evaluation of specific lysis and cytokine release, metabolomics and bioenergetics studies (using the hippocampus), intracellular FACS of cytokine production, ELISA spot assays, and lymphocyte subset analysis. Typically, a difference of approximately 2 to 3-fold in these assays may indicate a true biological difference. In some cases, differences of approximately 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or up to approximately 5 times, as measured in vitro after anti-GPC3 CAR immunoreactive cell therapy, can indicate therapeutic efficacy.

[0211] In some cases, subject CAR immunoreactive cell therapy can reduce tumor size by at least 30%, as the tumor size is measured by computed tomography (CT) scans or MRI. Anti-GPC3 CAR immunoreactive cell therapy can reduce tumor size by at least 30%.The size reduction is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100%. CAR-T therapy can eliminate tumors, as measured by CT scans. In some cases, anti-GPC3 CAR immune response cell therapy can effectively stabilize tumor size, where the change in tumor size is less than 10% from a baseline measurement of the diameter of the tumor lesion as measured by computed tomography (CT) scan. For example, the tumor size may not expand after administration of anti-GPC3 CAR immune response cells. In some cases, stability can be considered as a change in tumor size of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to pre-treatment measurements.

[0212] In some cases, the efficacy of anti-GPC3 CAR immune response cells can be considered based on subject survival time. For example, subjects treated with anti-GPC3 CAR immune response cells, such as anti-GPC3 CAR T cells, may survive longer than untreated subjects or subjects treated with different therapies.

[0213] In some cases, the efficacy of anti-GPC3 CAR immune response cells can be improved by adding secondary therapy, such as lymphopenic therapy. Secondary therapy may synergize with anti-GPC3 CAR immune response cell therapy. In some cases, secondary therapy may produce an additive effect of anti-GPC3 CAR immune response cell therapy. Secondary therapy may be lymphopenic therapy and other forms of cell therapy, antibody therapy, chemotherapy, radiotherapy, surgery, anti-angiogenic therapy, and any combination thereof.

[0214] Treatment response can be evaluated using the international criteria presented in the Revised Criteria for Evaluation of Treatment Efficacy in Solid Tumors (RECIST) guidelines (version 1.1). In the case of malignant lymph nodes, changes in the maximum diameter (one-dimensional measurement) and minimum diameter of the tumor lesion can be used in the RECIST criteria. For example, a measurable lesion can be defined as one that can be precisely measured in at least one dimension (the longest diameter to be recorded) as >20 mm (via chest X-ray), >10 mm (via CT scan), or >10 mm (via clinical examination using calipers). To be considered pathologically enlarged and measurable, a lymph node can be >15 mm on its short axis when assessed via CT scan. All other lesions (or disease sites), including small lesions (pathological lymph nodes with a longest diameter <10 mm or a short axis ≥10 to <15 mm), can be considered non-measurable. Bone lesions, meningeal diseases, ascites, pleural / pericardial effusions, cutaneous lymphangitis / pneumonia, and inflammatory breast diseases can be considered non-measurable.

[0215] All measurable lesions representing up to two per organ and up to five throughout the body can be...Target lesions are identified and recorded and measured at baseline. Target lesions may be selected based on their size (the lesion with the longest diameter), representing all relevant organs, but should otherwise be those suitable for repeatable measurements. It is possible that sometimes the largest lesion is not suitable for repeatable measurement; in this case, the second largest lesion that can be measured repeatedly should be selected. The sum of the diameters of all target lesions (the longest diameter for non-nodular lesions and the short axis for nodular lesions) can be calculated and reported as the baseline sum diameter. If the sum includes lymph nodes, only the short axis should be added to the sum. The baseline sum diameter will be used as a reference to page 28 / 55 of the specification, 31 CN 121102467 A, to further characterize any objective tumor regression in the measurable dimensions of the disease.

[0216] In some cases, when a clinical lesion may be superficial (e.g., skin nodules and palpable lymph nodes) and has a diameter of approximately 10 mm as assessed using calipers (e.g., skin nodules), the clinical lesion may be considered measurable. In cases where calipers cannot be used to measure the lesion, CT scans or MRI may be used. In some cases, CT scans may produce tissue sections of about 5 mm or smaller. In some cases, CT scans may have scan thicknesses of 5 mm, 4 mm, 3 mm, 2 mm, 1 mm, or 0.5 mm. If a CT scan has a section thickness greater than 5 mm, the minimum measurable lesion can be twice the section thickness. In some cases, MRI may also be performed to evaluate the subject. Ideally, the same type of scanner should be used, and the image acquisition protocol should be as close as possible to that of the previous scan when determining treatment efficacy. Where possible, a breath-hold scan technique should be used for body scanning. In some cases, fluorodeoxyglucose (FDG)-positron emission tomography (PET) may be used to measure treatment efficacy.

[0217] Once the subject has been evaluated, the target lesion may be classified as stable disease (SD), progressive disease (PD), partial response (PR), and / or complete response (CR). SD may be considered as the minimum sum of diameters that is neither sufficient to meet the reduction of PR nor sufficient to meet the increase of PD. PD can be considered as a minimum sum (which may include the baseline sum if it is possible to be minimum) with an increase of at least 20% in the total diameter of the target lesion. In some cases, in addition to a relative increase of about 20%, the sum must also show an absolute increase of at least about 5 mm. PR can be a reduction of at least about 30% in the total diameter of the target lesion with a reference to the baseline sum. CR can be the elimination of the target lesion.

[0218] In some cases, the combination of administration of anti-GPC3-CAR immune response cells (such as anti-GPC3 CAR T cells) and lymphopenia treatment can synergistically increase the median survival time of subjects compared to administration of anti-GPC3 immune response cells alone.Increase by at least about 6 months, Table 6. In some cases, the combination of anti-GPC3-CAR immune response cell therapy and lymphopenia treatment can synergistically increase the survival time of subjects by at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or up to about 25 years, compared with administration of anti-GPC3-CAR immune response cells alone. Table 6: Comparative Specification of Constructs 29 / 55 pages 32 CN 121102467 A

[0219] The subject anti-GPC3 CAR immune response cells can be formulated into medicines and used to treat human or mammalian patients diagnosed with a disease such as cancer. These medicines can be co-administered in human or mammalian patients with one or more chemotherapeutic agents or chemotherapeutic compounds.

[0220] Populations of subject CAR immune response cells, such as CAR T cells, can be formulated for administration to subjects using techniques known to those skilled in the art. Formulations containing CAR immune-response cell populations may contain pharmaceutically acceptable excipients. Excipients included in the formulation will serve different purposes, depending on, for example, the subset of T cells used and the administration method. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tensioning agents, fillers, and lubricants. Formulations containing CAR immune-response cell populations are typically prepared and cultured in the absence of any non-human components such as animal serum. A formulation may contain one CAR immune-response cell population, or more, such as two, three, four, five, six, or more CAR immune-response cell populations. For example, a formulation may contain one CAR T cell population, or more, such as two, three, four, five, six, or more CAR T cell populations.

[0221] Formulations containing anti-GPC3 CAR immune-response cell populations can be administered to subjects using methods and techniques known to those skilled in the art. Exemplary methods include, but are not limited to, intravenous injection. Other modalities include, but are not limited to, intratumoral, intradermal, subcutaneous (SC, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid region), intracranial, intraspinal, and intrathecal (spinal fluid). Any known device that can be used for parenteral injection or infusion of the formulation can be used to achieve such administration.

[0222] Formulations containing CAR immune response cell populations administered to subjects contain numerous therapeutic and / or preventative applications.CAR immune response cells effective for a specific indication or disease. Therefore, a therapeutically effective population of CAR immune response cells can be administered to the subject. Typically, a formulation containing approximately 1 x 10⁴ to approximately 1 x 10¹⁰ CAR immune response cells is administered. In most cases, the formulation will include approximately 1 x 10⁵ to approximately 1 x 10⁹ CAR immune response cells, approximately 5 x 10⁵ to approximately 5 x 10⁸ CAR immune response cells, or approximately 1 x 10⁶ to approximately 1 x 10⁷ CAR immune response cells. However, the number of CAR immune response cells administered to the subject will vary widely, depending on the location, origin, identity, extent and severity of the cancer, the age and condition of the individual to be treated, etc. The physician will ultimately determine the appropriate dose to be used.

[0223] Tumor-targeting molecules are administered to the subject before, simultaneously with, or after the administration of CAR immune response cells. Tumor-targeting molecules bind to target cells in the subject by associating with tumor-associated antigens or tumor-specific antigens. Tumor-targeting molecules can be formulated for administration to the subject using techniques known to those skilled in the art. Formulations of tumor-targeting molecules may contain pharmaceutically acceptable excipients. Examples of commonly used excipients include, but are not limited to: saline, buffered saline, dextran, water for injection, glycerol, ethanol and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tensioning agents, fillers and lubricants.

[0224] Tumor-targeting molecules may be administered to subjects using modalities and techniques known to those skilled in the art. Exemplary modalities include, but are not limited to, intravenous, intraperitoneal, and intratumoral injection. Other modalities include, but are not limited to, intradermal, subcutaneous (SC, sq, sub-Q, Hypo), intramuscular (IM), intraarticular, intramedullary, intracardiac, intra-articular (joint), intrasynovial (synovial fluid region), intracranial, intraspinal, and intrathecal (spinal fluid). In some cases, CAR-T may be administered locally at the site of a tumor lesion such as a liver lesion. Any known device that can be used for parenteral injection or infusion of the formulation may be used to achieve such administration.

[0225] A formulation containing a tumor-targeting molecule may be administered to a subject in an amount that is effective in treating and / or preventing a specific indication or disease. Typically, a formulation containing at least about 0.1 mg / kg body weight to about 100 mg / kg body weight of a tumor-targeting molecule is administered to the subject in need of treatment. In most cases, the dose is about 1 mg / kg to about 100 mg / kg body weight of the labeled protein daily, taking into account the route of administration, symptoms, etc. The physician will determine the appropriate dose to be used.

[0226] In one embodiment, the chimeric antigen receptor is used to stimulate an immune response mediated by immune response cells. For example, a T cell-mediated immune response is an immune response involving T cell activation. Activated antigen-specific cytotoxic T cells are able to induce apoptosis in target cells that display epitopes of foreign antigens on their surface, such as those displaying tumor antigens.Cancer cells. In another embodiment, a chimeric antigen receptor is used to provide anti-tumor immunity in mammals. The subject will develop anti-tumor immunity due to a T-cell-mediated immune response.

[0227] In some cases, a method of treating a subject with cancer may include administering to the subject in need of treatment an formulation of one or more tumor-targeting molecules, wherein these molecules bind to cancer cells, and administering one or more therapeutically effective subject-specific CAR immune-response cell populations, wherein the CAR immune-response cells can bind to the tumor-targeting molecules and induce cancer cell death. Another embodiment may relate to a method of treating a subject with cancer, which includes administering to the subject in need of treatment one or more therapeutically effective subject-specific anti-GPC3 CAR immune-response cell populations, wherein the CAR immune-response cells bind to cancer cells, thereby inducing cancer cell death.

[0228] The frequency of administration of both formulations comprising anti-GPC3 CAR immune-response cells and anti-GPC3 CAR immune-response cells in combination with tumor-targeting molecules will vary depending on factors including the disease being treated, the elements comprising CAR immune-response cells and tumor-targeting molecules, and the mode of administration. Each formulation may be administered independently four, three, two, or one time daily; once every other day, once every three days, once every four days, once every five days, once every six days, once a week, once every eight days, once every nine days, once every ten days, once every two weeks, once a month, and once every two months.

[0229] The terms “chemotherapeutic agent” or “chemotherapeutic compound” as used herein and their grammatical equivalents may refer to chemical compounds used in cancer treatment as described on pages 31 / 55 of CN 121102467 A. Chemotherapy cancer agents that may be used in combination with the disclosed CAR immune response cells include, but are not limited to, mitotic inhibitors (vinblastine alkaloids). These include vincristine, vinblastine, vindesine, and Navelbine™ (vinorelbine, 5'-nordehydrovinblastine). In other embodiments, cancer chemotherapeutic agents include topoisomerase I inhibitors, such as camptothecin compounds. As used herein, “camptothecin compounds” include Camptosar™ (irinotecan hydrochloride), Hycamtin™ (topotecan hydrochloride), and other compounds derived from camptothecin and its analogues. Another class of chemotherapeutic cancer agents that can be used in the methods and compositions disclosed herein are podophyllotoxin derivatives, such as etoposide, teniposide, and mitoxazoline. This disclosure also includes other chemotherapeutic cancer agents known as alkylating agents, which alkylate genetic material in tumor cells. These include, but are not limited to, cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphamide, carmustine, busulfan, chlorambucil, belustine, uramustine, chlomaphazin, and dacarbazine. This disclosure includes antimetabolites as chemotherapeutic agents.Examples of these types of agents include cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Another class of cancer chemotherapeutic agents that can be used in the methods and compositions disclosed herein include antibiotics. Examples include, but are not limited to, doxorubicin, bleomycin, actinomycin D, daunorubicin, styromycin, mitomycin C, and donomycin. Many commercially available liposomal formulations of these compounds are present. This disclosure also includes other cancer chemotherapeutic agents, including, but not limited to, antitumor antibodies, dacarbazine, azacytidine, acridine, melphalan, ifosfamide, and mitoxantrone.

[0230] Subject matter anti-GPC CAR immune response cells may be administered in combination with other antitumor agents, including cytotoxic / antitumor agents and antiangiogenic agents. Cytotoxic / antitumor agents can be defined as agents that attack and kill cancer cells. Some cytotoxic / antitumor agents can be alkylating agents, which alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylthiophosphamide, carmustine, busulfan, chlorambucil, belugutine, uramustine, chlomaphazin, and dacarbazine. Other cytotoxic / antitumor agents can be tumor cell antimetabolites, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine, and procarbazine. Other cytotoxic / antitumor agents can be antibiotics, such as doxorubicin, bleomycin, actinomycin D, daunorubicin, styromycin, mitomycin C, and donomycin. Many commercially available liposomal formulations of these compounds are present. Other cytotoxic / antitumor agents can be mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Other cytotoxic / antitumor agents include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, acridine, melphalan, VM-26, ifosfamide, mitoxantrone, and vincristine.

[0231] Anti-angiogenic agents may also be used. Suitable anti-angiogenic agents for the disclosed methods and compositions include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin-1 (including α and β), interleukin-12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and-2 (TIMP-1 and TIMP-2). Small molecules, including topoisomerases such as raszosen, and topoisomerase II inhibitors with anti-angiogenic activity, may also be used.

[0232] Other anticancer agents that can be used in combination with the subject anti-GPC3 CAR immune response cells include, but are not limited to: acivitine; doxorubicin; acodazole hydrochloride; acronin; adorine; interleukin; hexamethylmelamine; ampoxetine; and acrozin acetate.Anthraquinone; Aminoglutamine; Acridil; Anastrozole; Triamcinolone; Asparaginase; Tribulus terbinafine; Avastin; Azacitidine; Azatiprine; Azomycin; Balmastastat; Benzotiprine; Bicalutamide; Bismuth subsalicylate; Dinefadroxil; Bleomycin sulfate; Buquina sodium; Brompirimidine; Busulfan; Actinomycin C; Calotestosterone; Carbetamide; Carbetin; Carboplatin; Carmustine; Carrubicin Hydrochloride; Carcelexin; Sildenafil; Chlormethazine; Cisplatin; Cladribine; Clinapordine Mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin D; Daunorubicin Hydrochloride; Decitabine; Dextromethorphan; Dezaguanine Mesylate; Diazinon; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Instructions for Use 32 / 55 pages 35 CN 121102467 A Droloxifene Citrate; Drolbuterol Propionate; Dazomycin; Edatraxa; Eflunomide Hydrochloride; Exarucin; Enloplatin; Enpromethazine; Epilapidine; Epirubicin Hydrochloride; Ibuproazole; Exorubicin Hydrochloride; Estroxifene; Estroxifene Sodium Phosphate; Etamidazole; Etoposide; Etoposide phosphate; Etoposide; Fatrazol hydrochloride; Fazalabin; Fenivel Aamine; Fluorouracil; Fludarabine phosphate; Fluuracil; Flucitabine; Phosphorione; Fostracin sodium; Gemcitabine; Gemcitabine hydrochloride; Hydroxyurea; Idarubicin hydrochloride; Ifosfamide; Imofocin; Interleukin II (including recombinant interleukin II or rIL2); Interferon α-2a; Interferon α-2b; Interferon α-n1; Interferon α-n3; Interferon β-I; Interferon γ-Ib; Isopropylplatin; Irinotecan hydrochloride; Lanreotide acetate; Letrozole; Leuprorelin acetate; Riazoline hydrochloride; Lometraxo sodium; Lomustine; Loxoanthraquinone hydrochloride; Masrophenone; Metformin; Nitrogen mustard hydrochloride; Medroxyprogesterone acetate; Melenoprogesterone acetate; Melphalan; Minoril; Mercaptopurine; Methotrexate; Methotrexate sodium; Chlorpheniramine; Metformin; Mitocarcin; Mitocoline; Mitocin C; Mitospeptide; Mitotan; Mitoanthraquinone hydrochloride; Mycophenolic acid; Nocodazole; Nogamycin; Omaplatin; Oxysullen; Paclitaxel; Pegaspargase; Peripycin; Pendimethalin; Pelosinomycin Sulfate; Pephosphonamide; Piperabromide; Piperabromide; Pirroanthraquinone Hydrochloride; Procainamide; Promethene; Porphyrom sodium; Pofibromycin; Prednisone; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazosulfan; Lipoadenosine; Rogulam; Safungo; Safungosulfan Hydrochloride; Semustine; Citricazone; Sodium Phosphatidylcholine; Sparmycin; Germanium Spiroamide Hydrochloride; Spiromustine; Spiroplatin; Streptomycin; Streptozotocin; Sulfonamide; Tadalafil; Ticogalane Sodium; Tegafur; Teloxen HydrochlorideQuinone; Temopofen; Teniposide; Tiroxicon; Testrolide; Thiomipurine; Thioguanine; Thiotepa; Tiazofurin; Tirazamine; Toremifen citrate; Tritolon acetate; Tricerebin phosphate; Trimethotraxa; Trimethotraxa glucuronide; Triptorelin; Tobaccochloride hydrochloride; Uramustine; Uretepa; Vapotetide; Vertepofen; Vincristine sulfate; Vincristine sulfate; Vincristine sulfate; Vincristine sulfate; Vinpicidin sulfate; Vincristine sulfate; Vincristine sulfate; Vinrocin sulfate; Vinorelbine tartrate; Vinrodine sulfate; Vinridine sulfate; Vorticillium; Zonipram; Netostatin; Zorubicin hydrochloride. Other anticancer drugs include, but are not limited to: 20-epi-1,25-dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; ararubicin; acylfulren; adenocyclopentol; adorexin; adeleukin; ALL-TK antagonists; hexamethylmelamine; amimastatin; amidox; amifostine; aminolevulinic acid; ararubicin; acridine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antraloxetine; anti-dorsalizing morphogenetic protein-1; antiandrogens; prostate cancer; antiestrogens; antitumor ketones; antisense oligonucleotides; glycine afedipine; apoptosis gene regulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atametastan; amustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azamycin; diazotyrosine; berry gibberellin III derivative; balanol; balanol; CAR / ABL antagonist; benzodihydropyrrolidone; benzoyl astrosaponin; β-lactam derivative; β-alethine; β-clamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bistratene; diazidopropyltrimonium; nefenadine; bistratene A; breflate; brompirimidine; budotetan; sulfoxide imine; calcipotriol; calcium phosphate protein C; camptothecin derivative; canarypox IL-2; capecitabine; formamide-amino-triazole; carboxyamine-triazole; CaRest M3; CARN 700; chondroitin derivative inhibitors; cartilage-derived inhibitors; casein kinase inhibitors (ICOS); chestnut extract; silkworm antimicrobial peptide B; cetrorex; dihydroporphyrin; chloroquine sulfonamide; cisporin; cladribine; clomiphene analogues; clotrimazole; collimmycin A; collimmycin B; compressoritine A4; compressoritine analogues; conagenin; crambescidin816; Krestor; Nostocin 8; Nostocin A derivative; curacin A; cyclopentaquinone; cycloplatam; cypemycin; cytarabine octadecyl phosphate sodium; cytolysin; hexestrol phosphate; dacizumab; decitabine; dehydroepiandronate B; deselenoside; dexamethasone; dextromethorphan; dextromethorphan; dextromethorphan; dextromethorphan; dextromethorphan; diacin B; didox; diethyltocopherol; product manual 33 / 55 pages 36 CN 121102467 A Spermine; Dihydro-5-azacytidine; 9-Dihydrotamethasone; Dioxamycin; Diphenylspiromostatin; Docetaxel; Docetaxel; Dolastron; Deoxyfluorouridine; Droloxifen; Drocannabinol; Ducamycin SA; Ebuseline; Ekomustine; Edifosine; Edrecolomab; Eflunomide; Elimexene; Ethiributyrol; Epirubicin; Eprilate; Estrogen analogs; Estrogen agonists; Estrogen antagonists; Ethidazole; Etoposide phosphate; Exemestane; Fatrazol; Fazalabin; Fenivel Aamine; Filgrass; Finasteride; Vrappine; Flucalostemma; Fluasterone; Fludarabine; Fludorabin; Fludoramycin hydrochloride; Folicamin; Formexane; Forsitrocin; Formustine; Gadolinium texaphyrin; Gallium nitrate; Galotetabine; Ganirelix; Gelatinase inhibitor; Gemcitabine; Glutathione inhibitor; Hepsulfam; Modulin; Hexamethylene diacetamide; Hypericin; Ibandronic acid; Idarubicin; Edoxifen; Icaramone; Imofoxin; Ilomasta; Imidazolidine; Imiquimod; Immunostimulatory peptide; Insulin-like growth factor-1 receptor inhibitor; Interferon agonist; Interferon; Interleukin; Iodobenzylguanidine; Iododoxorubicin; 4-Sweet potato picrol; Iloprapane; Isopridine; Isobengazole; Isohomohalicondrin B; Itasetron; Jasplakinolide; Kahalalide F F); spirotin triacetate; lanreotide; ranamycin; levofloxacin; lentinan sulfate; leptolstatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprorelin + estrogen + progesterone; leuprorelin; levamisole; riazol; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; earthworm phospholipids; lometroxoline; chlordamine; loxoanthraquinone; lovastatin; loxoribin;Letotecan; lutetium texaphyrin; lysofylline; lysothelial peptide; methanotroph; manostatin A; marimastastatin; masorophorone; mammary filament inhibitor; matrix dissolving factor inhibitor; matrix metalloproteinase inhibitor; merbarone; avorelin; methionine; metoclopramide; MIF inhibitor; mifepristone; mitefocin; milistatin; mismatched double-stranded RNA; mitotoxin; dibromoceramide; mitomycin analogue; mitotoxin; mitotoxin fibroblast growth factor-saponin; mitotoxin; mofarotin; morastatin; human chorionic gonadotropin monoclonal antibody; monophospholipid A+ mycobacterium cell wall SK; mopidazole; multidrug resistance gene inhibitor; therapy based on multiple tumor suppressor gene 1; nitrogen mustard anticancer agent; Indian Ocean sponge B (mycaperoxide B); mycobacterium cell wall extract; myriaporone; N-Acetyldenafil; N-substituted benzamide; Nagrestip; Naloxone + Pentazocine; Napavin; Naphterpin; Natosine; Nedaplatin; Nemorubicin; Neridonic acid; Neutral endopeptidase; Nilumet; Nisamycin; Nitric oxide regulator; Nitrooxidant; Nitrullyn; O6-Benzylguanine; Octreotide; Ocitenone; Oligonucleotide; Ondansetron; Ondansetron; Oracin; Oral cytokine inducer; Omaplatin; Oxaliplatin; Oxalidone; Oxaunomycin; Paclitaxel; Paclitaxel analogs; Paclitaxel derivatives; Palolam (pa la ua) mine); palmitoyl raffinate; pamidronate; ginsenoside triol; panomiphen; parabactin; pozonipridine; pegasparginase; peldesine; pentosan polysulfate sodium; pentostatin; pentrazodazole; perfluorobromide; pephosphatamide; phenazinomycin; benzozidine; phenylacetate; phosphatase inhibitor; picibanil; pilocarpine hydrochloride; pirarubicin; pirarubicin; placetin A; placetin B; plasminogen activator inhibitors; platinum complexes; platinum compounds; platinum-triamine complexes; porphyrin sodium; pofibromycin; prednisone; propyl diacinone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; microalgal protein kinase C inhibitors; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; erythropoietin; pyrazoline acridine; pyridoxine-modified hemoglobin polyethylene oxide conjugates; RAF antagonists; raltitrexed; remoxicillinSetron; ras farnesyltransferase inhibitor; ras inhibitor; ras-GAP inhibitor; demethylated retepritin; rhenium etidronate Re 186; radicin; ribozyme; RII isotretinoin; roguimine; roxithoretin; romotide; roquimetac; rubiginone B1; ruboxyl; safingo; saintopin; SarCNU; inositol A; saxaglastine; Sdi 1 mimic; product information 34 / 55 pages 37 CN 121102467 A musstin; aging-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen-binding protein; cizonan; sobuzosen; boraxane; sodium phenylacetate; solverol; somatostatin-binding protein; sonaringin; phosphatidylcholine; spicamycin D; spiromostatin; snepandine; spongistatin 1 1); Squalamine; Stem cell inhibitor; Stem cell division inhibitor; Stipiamide; lysosome protease inhibitor; sulfinosine; Potent vasoactive intestinal peptide antagonist; Suradista; Suramin; Sorghum extract; Synthetic glycosaminoglycan; Tamustine; Tamoxifen methyl iodide; Tauromustine; Tazarotene; Tecogallan sodium; Tegafur; Tellurapyrylium; Telomerase inhibitor; Temopofol; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Bacterial embryogenesis; thiazolinone; thrombopoietin; thrombopoietin mimics; thymosin; thymopoietin receptor agonists; thymotrehnem; thyroid-stimulating hormone; ethyl cinnamate; teirazamine; timolocinolone dichloride; topsentin; toremifene; pluripotent stem cell factor; translation inhibitors; retinoic acid; triacetyluridine; trimethoprim; trimethoprim; triptorelin; tropisetron; tolostaniel; tyrosine kinase inhibitors; tyrosine phosphorylation inhibitors; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitors; urokinase receptor antagonists; valproic acid; variolin B; erythrocyte gene therapy vector systems; verarate; veratrine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; voroxetine; zanoteron; zeniplatin; benzyl vitamin C; and fenestrated succinate. In one embodiment, the anticancer drug is 5-fluorouracil, paclitaxel, or leucovorin.

[0233] In some cases, the subject anti-GPC3 CAR immune response cells can be introduced via injection, catheter, etc. In some cases, an immunostimulant may also be included, which includes, but is not limited to, interleukins (e.g., IL-2, IL-3, IL-6, and IL-11 and their derivatives).Interleukin), colony-stimulating factors (such as G-CSF, M-CSF, and GM-CSF), interferon (e.g., gamma-interferon), and erythropoietin. In some cases, subjects may be treated with CAR-T, immune-consuming agents, and immunostimulants. Subjects may be treated with IL-2 to enhance the performance of CAR-T cell products. In some cases, immunostimulants may be recombinant proteins. Immunostimulants may also include the active portion of a protein. In some cases, immunostimulants may include only a portion of a protein. A portion of a protein may be about 50%, 60%, 70%, 80%, 90%, or up to about 100% of the protein.

[0234] Compositions containing subject anti-GPC3 CAR immune response cells such as CAR T cells can be conveniently provided as sterile liquid formulations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions that can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are slightly easier to administer, particularly by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide a longer contact time with specific tissues. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.) and suitable mixtures thereof. As needed, sterile injectable solutions can be prepared by incorporating genetically modified CAR immune response cells for practicing the invention with various amounts of other desired components into a suitable solvent in the desired amount. Such compositions can be mixed with suitable carriers, diluents, or excipients such as sterile water, physiological saline, glucose, dextrose, etc. The compositions may also be lyophilized. The compositions may contain excipients such as wetting agents, dispersants, or emulsifiers (e.g., methylcellulose), pH buffers, gelling agents or viscosity-enhancing additives, preservatives, flavoring agents, colors, etc., depending on the route of administration and desired preparation. Suitable formulations can be prepared by referring to standard texts such as “REMINGTON'S PHARMACEUTICAL SCIENCE,” 17th edition, 1985, which is incorporated herein by reference, without extensive experimentation. Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of microbial action can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. Prolonged absorption of injectable drug forms can be achieved by using delayed absorption agents such as aluminum monostearate and gelatin. However, according to the invention, any carrier, diluent, or additive used must be compatible with genetically modified CAR immune response cells or their progenitor cells. (Instructions for Use, pages 35 / 55, 38 CN 121102467 A)

[0235] In some cases, the compositions may be isotonic, i.e., they may have the same osmotic pressure as blood and tears. The isotonicity required for the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextran, boric acid, sodium tartrate, propylene glycol, or other inorganic or organic solutes. Sodium chloride is preferred, especially for buffers containing sodium ions. If desired, a pharmaceutically acceptable thickener can be used to maintain the viscosity of the composition at a selected level. Methylcellulose is preferred because it is readily available and economical, and easy to use. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener will depend on the agent chosen. It is important to use an amount that will achieve the selected viscosity. Obviously, the choice of a suitable carrier and other additives will depend on the exact route of administration and the nature of the specific dosage form, such as a liquid dosage form (e.g., whether the composition will be formulated as a solution, suspension, gel, or another liquid form, such as a delayed-release or liquid-filled form).

[0236] In some cases, for example, in compositions, formulations and methods for treating cancer, the unit dose of the applied composition or formulation may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg. In some cases, the total amount of the composition or formulation applied may be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 g.

[0237] In some cases, the pharmaceutical composition comprising the subject anti-GPC3 CAR immune response cells such as CAR T cells may be administered alone or together with a pharmaceutically acceptable carrier or excipient via any route, and such administration may be in single or multiple doses. More specifically, the pharmaceutical composition can be combined with a variety of pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, sugar tablets, hand candy, powders, sprays, aqueous suspensions, injectable solutions, elixirs, syrups, etc. Such carriers include solid diluents or fillers, sterile aqueous media, and various non-toxic organic solvents. Furthermore, such oral pharmaceutical formulations can be suitably sweetened and / or flavored using various types of reagents commonly used for such purposes.

[0238] For example, cells can be administered together with any number of related treatments (e.g., before, simultaneously with, or after).Patients may receive treatments including, but not limited to, antiviral therapy, cidofovir and interleukin-2 or cytarabine (also known as ARA-C). In some cases, engineered cells may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunoablation agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytotoxins, fludaribine, cyclosporine, FK506, rapamycin, mycophenolate mofetil, steroids, FR901228, cytokines and radiation. Engineered cell compositions may also be administered to patients in conjunction with bone marrow transplantation and T-cell ablation therapies such as chemotherapy agents such as fludaribine, external beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH (e.g., before, during, or after). In some cases, engineered cell compositions may be administered after B-cell ablation therapy, which is an agent such as Rituxan that reacts with CD20. For example, a subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, after transplantation, the subject may receive an infusion of engineered cells, such as expanded engineered cells. Additionally, expanded engineered cells may be administered before or after the procedure. Engineered cells obtained by any of the methods described herein may be used for patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD). Therefore, methods for treating patients in need of treatment against host-vG rejection and graft-versus-host disease (GvHD) may be considered, including treating patients by administering an effective amount of genetically engineered cells containing inactivated TCRα and / or TCRβ. Kit Instructions 36 / 55 pages 39 CN 121102467 A

[0239] Kits containing compositions may be disclosed herein. This document also discloses kits for treating or preventing cancer, pathogen infections, immune disorders, or allogeneic transplantation. In one embodiment, the kit may contain a unit dose of a therapeutic or prophylactic composition containing an effective amount of cells, including one or more anti-GPC3 CARs. In some embodiments, the kit includes a sterile container that may contain a therapeutic or prophylactic vaccine; such a container may be a box, ampoule, vial, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing the drug. In some cases, the subject matter of anti-GPC3 CAR immune response cells, such as CAR T cells, may be related to the administration of CAR immune response cells to patients with cancer, pathogen infections, immune disorders, or allogeneic transplantation.The package insert is provided with the patient's information regarding the use of the composition for the treatment or prevention of cancer, pathogen infection, immune disorders, or allogeneic transplantation. In some cases, the kit may contain approximately 1 x 10⁴ cells to approximately 1 x 10¹² cells. In some cases, the kit may contain at least about 1 x 10⁵ cells, at least about 1 x 10⁶ cells, at least about 1 x 10⁷ cells, at least about 4 x 10⁷ cells, at least about 5 x 10⁷ cells, at least about 6 x 10⁷ cells, at least about 6 x 10⁷ cells, at least about 8 x 10⁷ cells, at least about 9 x 10⁷ cells, at least about 1 x 10⁸ cells, at least about 2 x 10⁸ cells, at least about 3 x 10⁸ cells, at least about 4 x 10⁸ cells, at least about 5 x 10⁸ cells, at least about 6 x 10⁸ cells, etc. At least approximately 6 x 10⁸ cells, at least approximately 8 x 10⁸ cells, at least approximately 9 x 10⁸ cells, at least approximately 1 x 10⁹ cells, at least approximately 2 x 10⁹ cells, at least approximately 3 x 10⁹ cells, at least approximately 4 x 10⁹ cells, at least approximately 5 x 10⁹ cells, at least approximately 6 x 10⁹ cells, at least approximately 6 x 10⁹ cells, at least approximately 8 x 10⁹ cells, at least approximately 9 x 10⁹ cells, at least approximately 1 x 10¹⁰ cells. At least about 2 x 10¹⁰ cells, at least about 3 x 10¹⁰ cells, at least about 4 x 10¹⁰ cells, at least about 5 x 10¹⁰ cells, at least about 6 x 10¹⁰ cells, at least about 6 x 10¹⁰ cells, at least about 8 x 10¹⁰ cells, at least about 9 x 10¹⁰ cells, at least about 1 x 10¹¹ cells, at least about 2 x 10¹¹ cells, at least about 3 x 10¹¹ cells, at least about 4 x 10¹¹ cells, at least about 5 x 10¹¹ cells, at least about 6 x 10¹¹ cells, at least about 6 x 10¹¹ cells, at least about 8 x 10¹¹ cells, at least about 9 x 10¹¹ cells, or at least about 1 x 10¹² cells. For example, a kit may contain about 5 x 10¹⁰ cells. In another example, the kit may contain 3 x 10⁶ cells; the cells may be expanded to approximately 5 x 10¹⁰ cells and administered to a subject.

[0240] In some cases, the kit may contain allogeneic cells. In some cases, the kit may contain cells that may have genomic modifications. In some cases, the kit may contain “ready-made” cells. In some cases, the kit may contain cells that can be expanded for clinical use. In some cases, the kit may contain contents for research purposes.

[0241] In some cases, the specification includes at least one of the following: a description of a therapeutic agent; for the treatment or prevention of tumors.Dosage and administration regimens for the causes, symptoms, or effects of pathogen infection, immune disorders, or allogeneic transplantation; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (if present), or as a label applied to the container, or as a separate sheet, brochure, card, or leaflet provided in or with the container. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells, such as anti-GPC3 CAR T cells, after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells before chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells concurrently with chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells at least 12 hours after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells at least approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, or up to 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after chemotherapy. In some cases, the instructions provide a procedure for administering anti-GPC3 CAR immune response cells at least 24 hours after chemotherapy. Anti-GPC3 CAR immune response cells can be formulated for intravenous injection. Anti-GPC3 CAR immune response cells can be formulated for intra-arterial injection into the liver of subjects who may contain solid tumors.

[0242] In some cases, the kit may contain cyclophosphamide and / or fludarabine, which are formulated for administration to subjects in need at about 60 mg / kg to about 80 mg / kg and about 25 mg / m2 to about 35 mg / m2, respectively. In some cases, the kit may contain pediatric doses of the product.

[0243] Recombinant methods are well known in the art. Unless otherwise stated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. Such techniques are well explained in references such as “Molecular Cloning: A Laboratory Manual,” 2nd edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (GaitEdited by Wei and Blackwell (1984); “Animal Cell Culture” (Edited by Freshney, 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (Edited by Wei and Blackwell); “Gene Transfer Vectors for Mammalian Cells” (Edited by Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Edited by Ausubel et al., 1987); “PCR: The Polymerase Chain Reaction” (Edited by Mullis et al., 1994); and “Current Protocols in Immunology” (Edited by Coligan et al., 1991). These techniques are applicable to the production of polynucleotides and peptides and can therefore be considered in the preparation and practice of the present invention. Particularly useful techniques are discussed in the following sections. Other Applications

[0244] Effective adoptive cell transfer-based immunotherapy (ACT) can be used to treat patients with cancer (e.g., metastatic cancer). For example, autologous peripheral blood lymphocytes (PBLs) can be modified using the non-viral or viral methods disclosed herein to express an anti-GPC3 chimeric antigen receptor (CAR) that recognizes GPC3 on cancer cells or tumor cells, and can be used in the disclosed methods and kits. The present invention relates to compositions and methods for use in immunotherapy (including, but not limited to, cancer therapy) using human or humanized chimeric antigen receptors after or concurrently with lymphopenic therapy in a subject. The chimeric antigen receptor utilizes a human or humanized chimeric antigen receptor construct after or concurrently with lymphopenic therapy in a subject.

[0245] These compositions and methods can provide cancer therapies with numerous advantages. In some cases, the methods may include modifying immune response cells so that the immune response cells are substantially independent of the presence or activity of the major histocompatibility complex (MHC). In some cases, the polynucleotides described herein may encode chimeric antigen receptors. Methods for preparing immune response cells expressing chimeric antigen receptors such as anti-GPC3 CARs are also disclosed. Treatment methods in which anti-GPC3 CAR immune response cells can be administered to a patient with cancer or disease.

[0246] This document describes methods for treating a disease (e.g., cancer) in a recipient, including transplanting one or more subject-specific anti-GPC3 CAR immune-response cells into the recipient following or simultaneously with lymphopenia treatment.

[0247] Autologous lymphocyte infusion can be used for treatment. Autologous peripheral blood mononuclear cells (PBMCs) can be collected from a patient in need of treatment, and T cells can be activated and expanded using methods described herein and known in the art, and then infused back into the patient. In other cases, allogeneic cells can be used to treat the patient. Subject CAR immune response cell populations can be formulated for administration; and the subjects are administered the cells using techniques known to those skilled in the art. The expanded cells can then be grown under conditions similar to those for unmodified cells, thereby allowing the modified cells to be expanded and used for various purposes.

[0248] The methods disclosed herein may include transplantation. Transplantation can refer to adoptive transplantation of cell products. Transplantation can be autologous, allogeneic, xenogeneic, or any other type of transplantation. For example, transplantation can be xenogeneic. Transplantation can also be allogeneic. Specification 38 / 55 pages 41 CN 121102467 A

[0249] In some cases, approximately 5 x 10¹⁰ subject anti-GPC3 CAR immune response cells are administered to the subject. In some implementations, approximately 5 x 10¹⁰ cells represent the median number of cells administered to the subject. In some implementations, approximately 5 x 10¹⁰ cells are necessary to influence the subject's treatment response. In some implementations, at least approximately 1 x 10⁷ cells, at least approximately 2 x 10⁷ cells, at least approximately 3 x 10⁷ cells, at least approximately 4 x 10⁷ cells, at least approximately 5 x 10⁷ cells, at least approximately 6 x 10⁷ cells, at least approximately 7 x 10⁷ cells, at least approximately 8 x 10⁷ cells, at least approximately 9 x 10⁷ cells, at least approximately 1 x 10⁸ cells, at least approximately 2 x 10⁸ cells, at least approximately 3 x 10⁸ cells, at least approximately 4 x 10⁸ cells, at least approximately 5 x 10⁸ cells, etc. At least approximately 6 x 10⁸ cells, at least approximately 7 x 10⁸ cells, at least approximately 8 x 10⁸ cells, at least approximately 9 x 10⁸ cells, at least approximately 1 x 10⁹ cells, at least approximately 2 x 10⁹ cells, at least approximately 3 x 10⁹ cells, at least approximately 4 x 10⁹ cells, at least approximately 5 x 10⁹ cells, at least approximately 6 x 10⁹ cells, at least approximately 7 x 10⁹ cells, at least approximately 8 x 10⁹ cells, at least approximately 9 x 10⁹ cells. At least approximately 1 x 10¹⁰ cells, at least approximately 2 x 10¹⁰ cells, at least approximately 3 x 10¹⁰ cells, at least approximately 4 x 10¹⁰ cells, at least approximately 5 x 10¹⁰ cells, at least approximately 6 x 10¹⁰ cells, at least approximately 7 x 10¹⁰ cells, at least approximately 8 x 10¹⁰ cells, at least approximately 9 x 10¹⁰ cells, at least approximately 1 x 10¹¹ cells, at least approximately 2 x 10¹¹ cells, at least approximately 3 x 10¹¹ cells, at least approximately 4 x 10¹¹ cells, at least approximately 5 x 10¹¹ cells, at least approximately 6 x 10¹¹ cells, at least approximately 7 x 10¹¹ cells, at least approximately8 x 10¹¹ cells, at least about 9 x 10¹¹ cells, or at least about 1 x 10¹² cells. For example, about 5 x 10¹⁰ cells may be administered to a subject. In another instance, cells may be expanded from 3 x 10⁶ cells to about 5 x 10¹⁰ cells and administered to a subject. In some cases, cells are expanded to a number sufficient for therapeutic use. For example, 5 x 10⁷ cells may undergo rapid expansion to produce a number sufficient for therapeutic use. In some cases, a number sufficient for therapeutic use may be 5 x 10¹⁰. Any number of cells may be infused for therapeutic use. For example, a subject may be infused with cell numbers from 1 x 10⁶ to 5 x 10¹² (inclusive). Patients may be infused with as many cells as can be produced for them. In some cases, not all cells infused into a patient are engineered. For example, at least 90% of the cells infused into a patient may be engineered. In other cases, at least 40% of the cells infused into a patient may be engineered. In some embodiments, the method may include calculating the amount of engineered cells necessary to influence a treatment response in a subject and / or administering them to the subject. In some embodiments, calculating the amount of engineered cells necessary to influence a treatment response includes cell viability and / or the efficiency with which the anti-GPC3 CAR transgene has been integrated into the cell's genome. In some embodiments, the cells administered to the subject to influence a treatment response may be live cells. In some embodiments, to achieve a treatment response in a subject, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, and at least about 10% of the cells are live cells. In some embodiments, to influence a treatment response in a subject, the cells administered to the subject may be cells having one or more transgenes successfully integrated into the cell's genome. In some embodiments, in order to achieve a therapeutic response in a subject, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, and at least about 10% of the cells have one or more CAR transgenes successfully integrated into the cell genome.

[0250] In some cases, subject-specific anti-GPC3 CAR immune response cells may be administered to the subject, wherein the administerable CARImmune response cells can range in size from approximately 1 day to approximately 35 days. For example, the cells administered can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or up to 40 days old. The age of CAR immune response cells can be considered from the time of stimulation. The age of CAR immune response cells can be considered from the time of blood component apheresis. The age of CAR immune response cells can be considered from the time of transduction. In some implementation instructions (pages 39 / 55, CN 121102467 A), the CAR immune response cells that can be administered to the subject are approximately 10 to approximately 14 days or approximately 20 days old. In some cases, the "age" of CAR immune response cells can be determined by the length of telomeres. For example, “young” CAR immune response cells may have longer telomere lengths than “consumed” or “old” CAR immune response cells. Without being bound by any particular theory, it can be assumed that immune response cells lose approximately 0.8 kb of telomere length per week in culture, and that young CAR immune response cell cultures may have telomeres approximately 1.4 kb longer than those of immune response cells approximately 44 days old. Without being bound by any particular theory, it is assumed that longer telomere lengths may be associated with a positive objective clinical response in patients and cell persistence in vivo.

[0251] In some cases, cells are isolated from a subject organism, transfected with nucleic acids (e.g., genes or cDNA), and re-infused into the subject organism (e.g., a patient).

[0252] Cells (e.g., engineered cells or engineered primary T cells) may function before, after, and / or during transplantation. For example, transplanted cells may function for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days after transplantation. Transplanted cells may function for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after transplantation. Transplanted cells may function for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 years after transplantation. In some cases, transplanted cells may function for up to the recipient's lifetime.

[0253] Furthermore, transplanted cells may perform 100% of their normal expected function. The transplanted cells can also perform their normal expected functions at rates of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15%.16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or up to about 100%.

[0254] The transplanted cells may also perform more than 100% of their normal expected function. For example, the transplanted cells may perform 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or up to about 5000% of their normal expected function.

[0255] Transplantation can be performed by any type of transplant. Sites may include, but are not limited to, the subcapsular space of the liver, the subcapsular space of the spleen, the subcapsular space of the kidney, the omentum, the submucosa of the stomach or intestine, segments of small intestinal vessels, venous sacs, the testes, the brain, the spleen, or the cornea. For example, the transplant may be a subcapsular transplant. The transplant may also be an intramuscular transplant. The transplant may be an intraportal transplant.

[0256] Following treatment (e.g., any treatment disclosed herein), transplant rejection may be improved compared to transplanting one or more wild-type cells into the recipient. For example, transplant rejection may be hyperacute rejection. Transplant rejection may also be acute rejection. Other types of rejection may include chronic rejection. Transplant rejection may also be cell-mediated rejection or T-cell-mediated rejection. Transplant rejection may also be natural killer cell-mediated rejection.

[0257] Improved transplantation may mean mitigating hyperacute rejection, which may include a reduction, mitigation, or attenuation of adverse effects or symptoms. Transplantation may refer to adoptive transplantation of cell products.

[0258] Another indicator of successful transplantation may be the number of days the recipient does not require immunosuppressive therapy. For example, following the treatments provided herein (e.g., transplantation), the recipient may not require immunosuppressive therapy for at least or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. This can indicate that the transplantation was successful. It can also indicate that the transplanted cells, tissues, and / or organs were not rejected.

[0259] In some cases, the recipient may not require immunosuppressive therapy for at least 1 day. The recipient may not require immunosuppressive therapy for at least 7 days. The recipient may not require immunosuppressive therapy for at least 14 days. The recipient may not require immunosuppressive therapy for at least 21 days. The recipient may not require immunosuppressive therapy for at least 28 days. The recipient may not require immunosuppressive therapy for at least 60 days. Furthermore, the recipient may not require immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0260] Another indicator of a successful transplant may be the number of days the recipient requires reduced immunosuppressive therapy. For example, following the treatments described herein, the recipient may require reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days or more. This may indicate that the transplant is successful. This may also indicate that the transplanted cells, tissues, and / or organs have no rejection or minimal rejection.

[0261] In some cases, the recipient may require reduced immunosuppressive therapy for at least 1 day. The recipient may also require reduced immunosuppressive therapy for at least 7 days. The recipient may require reduced immunosuppressive therapy for at least 14 days. The recipient may require reduced immunosuppressive therapy for at least 21 days. The recipient may require reduced immunosuppressive therapy for at least 28 days. The recipient may require reduced immunosuppressive therapy for at least 60 days. Furthermore, the recipient may require reduced immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more.

[0262] Reduced immunosuppressive therapy may refer to less immunosuppressive therapy compared to the immunosuppressive therapy required when transplanting one or more wild-type cells into the recipient.

[0263] Immunosuppressive therapy may include any treatment that suppresses the immune system. Immunosuppressive therapy can help alleviate, minimize, or eliminate transplant rejection in the recipient. For example, immunosuppressive therapy may include immunosuppressive drugs. Immunosuppressive drugs that can be used before, during, and / or after transplantation are not limited to MMF (mycophenolate mofetil (Cellcc)), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alenzusmab (Campath), anti-CD20 (rituximab), anti-IL-6R antibody (tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab), CTLA4-Ig (abatacept / Orencia), beracipex (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), dalizumab (Ze-napax), and baliximab.Simulect, infliximab (Remicade), cyclosporine, deoxyguanidine, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, and anti-CD122 antibody. In addition, one or more immunosuppressants / drugs may be used together or sequentially. One or more immunosuppressants / drugs may be used for induction therapy or maintenance therapy. The same or different drugs may be used in the induction and maintenance phases. In some cases, dalizumab (Zenapax) may be used for induction therapy, while tacrolimus (Prograf) and sirolimus (Rapimune) may be used for maintenance therapy. Dalizumab (Zenapax) can also be used for induction therapy, while low-dose tacrolimus (Prograf) and low-dose sirolimus (Rapimune) can be used for maintenance therapy. Immunosuppression can also be achieved using non-pharmacological regimens, including but not limited to whole-body irradiation, thymic irradiation, and total and / or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs. Examples

[0264] The invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the invention should never be construed as limited to the following examples, but should be construed as covering any and all variations that become apparent from the teachings provided herein. Example 1: Construction of an anti-GPC3 CAR vector

[0265] An exemplary lentiviral plasmid vector was constructed using a third-generation self-inactivated lentiviral vector system. The system comprises a packaging plasmid pMDLg RRE (Addgene) encoding Gag / Pol, a packaging plasmid pRSV-REV (Addgene) encoding Rev, an envelope plasmid pCMV-VSV-G (Addgene) encoding VSV-G, and a recombinant expression vector encoding the CAR gene based on the empty vector pRRLSIN-cPPT.PGK-GFP.WPRE (Addgene). This system effectively reduces the risk of forming replicative lentiviral (RCL) particles.

[0266] The empty vector pRRLSIN-cPPT.PGK-GFP.WPRE contains a promoter for elongation factor-1α (EF-1α), and an MluI cleavage site is inserted between this promoter and the CD8αsp signal peptide. Specifically, it is double-digested with ClaI / SalI (NEB).The pWPT-EGFP vector (Addgene) was used to re-obtain a 1.1 kb DNA fragment, which was then ligated with T4 DNA ligase to pRRLSIN-cPPT.PGK-GFP.WPRE, which was double-digested with ClaI / SalI (NEB). The ligation mixture was then transformed into TOP10 host cells. Positive clones were identified by colony PCR and confirmed by sequencing, yielding the recombinant plasmid pRRLSIN-cPPT.EF-1α-EGFP.WPRE. The EF-1α promoter (SEQ ID NO: 33, containing the Mlu I cleavage site) of the CD8α signaling domain (also known as fragment 1, 442 bp) was amplified using upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 31), downstream primer 5'-CGGCCTGGCGGCGTGGAG-3' (SEQ ID NO: 32), and pRRLSIN-cPPT.EF-1α-EGFP.WPRE as a template. Amplification was performed under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 68 °C for 30 s; and after 25 cycles, extension at 68 °C for 10 min. Agarose gel electrophoresis confirmed that the amplified bands had the expected fragment size.

[0267] Humanized antibody 35 has been described in Chinese patent application number CN201510481235.1, which is capable of specifically recognizing humanized GPC3 protein. To construct the 35-CAR lentiviral plasmid, the following conditions were used during amplification to provide the heavy chain variable domain fragment: a fragment containing the heavy chain variable domain 35 (SEQ ID NO: 80 in CN201510481235.1) was used as a template, with upstream primer 5'-ctccacgccgccaggccggaggtgcagctggtgcag-3' (SEQ ID NO: 34) and downstream primer 5'-GCGGTGTCCTCGCTCCGCAGGCTGCTCAGCTCCATGTAGGCGTG-3' (SEQ ID NO: 35); the following conditions were used to provide the light chain variable domain fragment: a fragment containing the light chain variable domain 35 (SE QID NO: 79, CN201510481235.1) was used as a template, with upstream primer 5'-GCGGAGCGAGGACACCGCCGTGTACTACTGCGC CCGGTTCT ACAGCTAC-3' (SEQ ID NO: 80) NO:36), and downstream primer 5'-CGGCGCTGGCGTCGTGGTACGTTTGA TCTCCAGCTTGGTG-3' (SEQ ID ...).(SEQ ID NO:37). Bridged PCR was used to amplify the variable domain primers of the heavy and light chains to provide a 35scFv fragment (SEQ ID NO:38, also known as fragment 2, 765 bp) containing sequences repeating the upstream CD8α signal peptide and the downstream hinge region. PCR amplification was performed under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s, annealing at 58 °C for 40 s; extension at 68 °C for 40 s; and after 25 cycles, extension at 68 °C for 10 min. The amplified bands were confirmed to be of the expected fragment size by agarose gel electrophoresis.

[0268] Using upstream primer 5'-accacgacgccagcgccg-3' (SEQ ID NO:39) and downstream primer 5'-aatcc agaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO:40), pWPT-eGFP-F2A-GPC3-28Z was used as a template (see Chinese patent application CN 104140974 A) to amplify Hinge-28Z (SEQ ID NO:41, fragment 3, 703bp) (with an internal Sal I cleavage site). PCR amplification was performed under the following conditions: initial denaturation at 94°C for 4 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s; extension at 68°C for 30 s; and a final extension at 68°C for 10 min after 25 cycles. The amplified bands were confirmed to have the expected fragment size by agarose gel electrophoresis. Equimolar amounts (approximately 50 ng) of fragments 1, 2, and 3 were amplified by overlap PCR under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s; extension at 68 °C for 140 s; and a final extension at 68 °C for 10 min after 5 cycles. The mixture was then supplemented with DNA polymerase, upstream primer 5'-gcaggggaaagaatagtagaca-3' (SEQ ID NO: 31), and downstream primer 5'-aatccagaggttgattgtcgacctagcgagggggcagggcctgc-3' (SEQ ID NO: 40), and amplified for 25 cycles under the following conditions: initial denaturation at 94 °C for 4 min; denaturation at 94 °C for 40 s; annealing at 60 °C for 40 s; extension at 68 °C for 140 s; and a final extension at 68 °C for 10 min. The theoretical size of the obtained 35-28Z (SEQ ID NO:41) is 1874 bp. The amplified product was confirmed by agarose gel electrophoresis and has the expected fragment size, as shown in SEQ ID NO:23 on pages 42 / 55 of the sequence specification, CN 121102467 A.

[0269] The vector pRRLSIN-cPPT.EF-1α-EGFP.WPRE and 35-28Z were digested with MluI and SalI, then ligated with T4 ligase and transformed into TOP10. Positive clones were identified by colony PCR and confirmed by Invitrogen sequencing, confirming the acquisition of pRRL-EF-1α-35-28Z. Example 2: Packaging of anti-GPC3-CAR lentivirus

[0270] Anti-GPC3-CAR lentivirus stock solution (0.75L) was prepared using embryonic kidney cells 293T (ATCC: CRL-11268). Specifically, 293T cells were seeded at 1.17×10⁴ / cm² on 5 layers of cellstack (Corning). On the day of transfection, solution A was prepared containing 133.2 μg of packaging plasmid pRSV-Rev, 133.2 μg of pMDLg / pRRE, 51.56 μg of pCMV-VSV-G, and 111.7 μg of pRRL-EF-1α-35-28Z. This was achieved by dissolving 966 μg of all plasmids in 17 ml of DMEM basal medium and mixing gently. Solution B was prepared by dissolving 2.9 mg of PEI (Polysciences) in 17 ml of DMEM basal medium, mixing gently, and incubating at room temperature for 5 min. Solution A was then added to solution B, mixed thoroughly, and allowed to stand at room temperature for 20-25 min. The 293T culture was then transferred from the Cellstack to a 1 L flask. The combined solution containing the plasmids and PEI was then added to the culture solution and mixed gently. The resulting solution was then transferred to a Cellstack and incubated at 37 °C and 5% CO2 for 5 h. The culture medium was then replaced with fresh growth medium DMEM supplemented with 10% FBS (Life Technology). The culture was then incubated at 37 °C and 5% CO2 for 48 h. The lentivirus stock solution was then obtained again, filtered through a 0.45 μm (Millipore) filter, concentrated, purified using a IIi tangential flow filtration system (Spectrum), washed with AIM-V (Life Technology), and stored. Virus titration was performed according to the method described in CN104140974A. The virus titer was calculated to be approximately 1 × 10⁸ / ml. The virus was packaged at 1 × 10⁸ / vial and stored at -80 °C for subsequent use. Example 3: Preparation of anti-GPC3-CAR T cells and in vitro antitumor assay against liver cancer cells

[0271] CAR T cells were prepared in an immune cell preparation laboratory according to cGMP standards. Specifically, peripheral blood or leukocyte-enriched blood obtained from subjects using the COBE Spectra blood component separator was separated by density gradient centrifugation.Human peripheral blood mononuclear cells (PBMCs) were thus obtained. PBMCs were mixed with magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Life Technology) at a 1:3 PMBC to magnetic bead ratio, stimulated with recombinant human IL-2 (Shanghai Hua Xin High Biotechnology Co., Ltd.) at a final concentration of 300 U / mL, and cultured for 48 h. Subsequently, activated T cells were transduced using the aforementioned lentivirus at approximately 5 MOI, with transduction efficiency enhanced by RetroNectin (Takara). At 24 h and 48 h post-transduction, the culture medium was changed by low-speed centrifugation (100 g × 10 min) to remove free virus. Magnetic beads were removed at 72 h post-transduction. The above centrifugation was repeated, and passaged cultures at a density of 5 x 10⁵ / mL were obtained. Subsequently, cells were passaged every other day at a density of approximately 3 x 10⁵ / mL, while recombinant human IL-2 was added to the lymphocyte culture medium at a final concentration of 300 U / mL.

[0272] After culturing in vitro for 10 to 12 days, the CAR expression of transduced CAR T cells was determined by flow cytometry. In other words, the positive transduction rate of CAR T cells was determined, and it is shown in Table 7. In addition, in vitro antitumor assays were performed by co-incubating with high GPC3-expressing hepatocellular carcinoma cells Huh-7, low GPC3-expressing PLC / PRF / 5, and GPC3-negative SK-HEP-1 for 18 h, with effector-to-target ratios of 3:1, 1:1, and 1:3. Detailed procedures are described in CN104140974A. Table 8 presents the in vitro antitumor activity results at an effector-to-target ratio of 1:1.

[0273] The results showed that approximately 40% to approximately 80% of the anti-GPC3-CAR T cells expressed CAR. In addition, anti-GPC3-CAR T cells specifically killed GPC3-positive Huh-3 and PLC / PRF / 5, with a dose-dependent effector:target ratio, while they had no killing effect on GPC3-negative SK-HEP-1 (Table 8), indicating that the anti-GPC3-CAR T cells prepared by the subjects in this disclosure have excellent targeted killing effects.

[0274] Table 7: Overview of positive transduction rate and administration regimen of anti-GPC3-CAR T cells from subjects 43 / 55 pages 46 CN 121102467 A

[0275] Table 8: In vitro killing of anti-GPC3-CAR T cells from subjects (effector:target ratio of 3:1, 1:1 and 1:3, in vitro culture for 18h) Example 4: Changes in lymphocytes after lymphopenia

[0276] After confirming that no abnormalities occurred during the preparation of anti-GPC3-CAR T cells by the subjects, various physical conditions of the subjects were investigated.A comprehensive assessment was conducted to determine whether the subject was suitable for in vivo lymphopenia treatment. A primary lymphopenia regimen was designed for use in suitable subjects.

[0277] The following two lymphopenia regimens were designed: Regimen 1: Cyclophosphamide (CTX) monotherapy lymphopenia regimen, dose 1 g / m2 / day x 1 day. Regimen 2: Fludarabine (FLU) + cyclophosphamide combination lymphopenia regimen, dose 20-30 mg fludarabine / m2 / day x 4 days + 500 mg cyclophosphamide / m2 / day x 2 days.

[0278] The regimen was implemented with adjustments including the following conditions: the human body surface area was calculated according to the Stevenson formula (Chinese manual 44 / 55 pages 47 CN 121102467 A Journal of Physiology, 12:327, 1937). That is, surface area (m2) = 0.0061 x height (cm) + 0.0128 x weight (kg) - 0.1529. For example, an adult with a height of 170cm and a weight of 60kg has a surface area of ​​1.6521m2. Subjects who were not suitable for lymphopenia treatment due to their existing physical condition or their own choice were assigned to the second cohort. A total of three subjects (H01, H02, and H03) received different lymphopenia treatments. One subject (H04) was not ablated due to his physical condition and his own choice. The protocols are shown in Table 9.

[0279] Table 9: Overview of lymphopenia protocols for subjects

[0280] H011 indicates the initial administration of anti-GPC3-CAR T-cell therapy to subject H01; H012 indicates the second administration of anti-GPC3-CAR T-cell therapy to subject H01; and D0 is defined as the day of administration of anti-GPC3-CAR T-cell therapy. Following the implementation of the above-described lymphopenia regimen, various changes occurred in the absolute lymphocyte counts of the subjects. Lymphopenia treatment with cyclophosphamide monotherapy resulted in a lymphopenia reduction of approximately 40% to approximately 72%. Lymphopenia treatment with the combination of fludarabine and cyclophosphamide produced a more significant difference in lymphopenia reduction compared to cyclophosphamide monotherapy, ranging from approximately 82% to approximately 96.5%. Specifically, Figure 2 shows the changes in the ratio of absolute lymphocyte counts to baseline at each monitoring time point before and after the administration of lymphopenia treatment. These results demonstrate that the combination of fludarabine and cyclophosphamide results in superior lymphopenia compared to the cyclophosphamide monotherapy regimen. Example 5: Clinical Response of Subjects After Ablation

[0281] Approximately 2 days after the implementation of lymphopenia treatment, subjects were intravenously administered anti-GPC3-CAR T-cell therapy. The administered anti-GPC3-CAR...The T-cell dosages are presented in Table 7. A summary of the clinical responses of the subjects after treatment is shown in Table 10. Results demonstrate that, to date, the three subjects who received anti-GPC3-CAR T-cell therapy for lymphopenia have stable disease (SD) or partial disease (PR). Conversely, subject H04, who did not receive lymphopenia treatment, showed progressive disease on MRI 4 weeks after anti-GPC3-CAR T-cell therapy.

[0282] Regarding AFP results, due to the insensitivity of AFP, the prognosis of subject H02 could not be assessed from tumor markers. For the other two subjects, their AFP levels decreased slightly, as shown in Table 10. It should be noted that subject H03 showed an 87% reduction in AFP after lymphopenia treatment with a combination of fludarabine and cyclophosphamide compared to baseline before treatment. Simultaneously, imaging showed partial clinical response at the target site. Figure 3 shows the MRI results of subject H03 before treatment and 10 weeks after treatment.

[0283] Table 10: Overview of Clinical Responses in Subjects After Anti-GPC3-CAR T-Cell Therapy (Instructions for Use, Pages 45 / 55, 48, CN 121102467 A)

[0284] Note: ↓ indicates a decrease in AFP levels; ↑ indicates an increase in AFP levels; PD indicates progressive disease; CR indicates a complete response; and PR indicates a partial response. Safety data and clinical observations showed that no subjects experienced intolerable toxicity or side effects after treatment with anti-GPC3-CAR T-cells. All subjects experienced some degree of fever and increased GRP. Subjects H01 and H04 experienced shivering after administration. Subject H03 had a decrease in albumin due to fever. By ingesting exogenous nutrients and albumin, the subject had normal albumin levels after the fever subsided (Table 11).

[0285] Table 11: Overview of Toxicity or Side Effects in Subjects After Administration of Anti-GPC3-CAR T-Cells Following Lymphopenia Treatment

[0286] Furthermore, no cytokine release syndrome, which frequently occurs during CAR T-cell therapy for hematologic malignancies, was observed in this clinical study, indicating that anti-GPC3-CAR T cells are safe.

[0287] In summary, the clinical study results of CAR-GPC T-cell therapy for GPC3-positive hepatocellular carcinoma demonstrate that subjects who received effective lymphopenic therapy with anti-GPC3-CAR T-cell therapy gained some clinical benefit, while subjects who did not receive lymphopenic therapy did not gain any clinical benefit. Furthermore, neither subjects who received nor did not receive lymphopenic therapy exhibited intolerable toxicity or side effects; both had certain adverse reactions, such as fever and increased CRP, suggesting that lymphopenic therapy may not have exacerbated adverse reactions in the subjects. In addition,In terms of the efficacy of lymphopenic therapy and the benefits to subjects, the combined fludarabine and cyclophosphamide lymphopenic purpura regimen may be superior to the cyclophosphamide monotherapy lymphopenic purpura regimen. In summary, anti-GPC3-CAR T-cell therapy following effective lymphopenic purpura treatment can provide an effective treatment solution for the clinical treatment of GPC3-positive hepatocellular carcinoma. Example 6: Phenotypic Analysis

[0288] The expression of cell surface molecules was determined by flow cytometry using standard methodologies. The following monoclonal antibodies conjugated to phycoerythrin, fluorescein isothiocyanate, and / or polydinophytin chlorophyll protein were used: CD3, CD4, CD8, CD30, CCR4, CD45RA, CD45RO, CCR7, CD62L, CD56, αβ T-cell receptor (BD Biosciences PharMingen). CAR expression on T cells was detected using anti-CAR antibodies, according to the product manual, pages 46 / 55, 49 CN 121102467 A. Samples were analyzed using FACSCalibur (BD Biosciences PharMingen), and data were analyzed using CellQuest Pro software (BD Biosciences, San Jose, CA). At least 10,000 positive events were measured for each sample. Example 7: Cytotoxicity Assay: Hepatocellular Carcinoma

[0289] To assess the potential cytotoxic effects of second- or third-generation CAR-T transduced T cells, a CytoTox non-radioactive cytotoxicity assay (Promega) was performed. Briefly, anti-GPC3 CAR-T cells (33-28 BBZ, 92-28 BBZ, and 4-28 BBZ) were co-cultured with control cells SK-HEP-1 (Fig. 4A) and CHO-K1 (Fig. 4B) at an effector:target ratio of 3:1, 1:1, or 1:3, with 10,000 target cells per well, and incubated at 37°C for 18 hours. Visible absorbance data were collected after incubation using a standard 96-well plate reader.

[0290] To evaluate antitumor cytotoxicity, CAR-T cells 33-28BBZ, 92-28BBZ, and 4-28BBZ were co-cultured with GPC3-positive hepatocellular carcinoma cells Huh-7 or control cells transduced to express GPC3, CHO-K1-GPC3+. The effector:target ratio was 3:1, 1:1, or 1:3, with 10,000 target cells per well. Cells were incubated at 37°C for 18 hours (Figures 5A and 5B, respectively). Visible absorbance data were collected after incubation using a standard 96-well plate reader.

[0291] For all effector:target ratios, cytotoxicity was calculated as the average of five replicate wells. Results

[0292] Cytotoxicity data showed that at 3:1, 1:1, and 1:3, 33-28BBZ, 92-28BBZ, and 4-28BBZ CAR-T cells exhibited good antitumor cytotoxicity.The construct exhibited cytotoxicity against tumor cell lines expressing GPC3, but not against control lines not expressing GPC3. Example 8: Cytotoxicity assay: Hepatocellular carcinoma group

[0293] To evaluate the potential cytotoxic effects of second- or third-generation CAR-T transduced T cells, a CytoTox non-radioactive cytotoxicity assay (Promega) was performed. Briefly, anti-GPC3 CAR-T cells 92-28Z, 92-BBZ, 92-28BBZ or empty vector were co-cultured with HepG2 (HCC) (Figure 6A), Hep3B (HCC) (Figure 6B) or PLC / PRF / 5 (hepatocellular carcinoma) (Figure 6C) at an effector:target ratio of 3:1, 1:1 or 1:3, with 10,000 target cells / well, and incubated at 37°C for 18 hours. To evaluate the cytotoxic effects of second- and third-generation CAR-T cells, similar CytoTox assays were performed using 92-28Z, 92-BBZ, 92-28BBZ, or empty vectors co-cultured with Huh-7 hepatocellular carcinoma cells with high GPC3 expression (Figure 7A). Visible absorbance data were collected after incubation using a standard 96-well plate reader. Results

[0294] Cytotoxicity data showed that the second- and third-generation constructs exhibited comparable cytotoxicity in all hepatocellular carcinoma groups, including HCC and hepatocellular carcinoma cell lines, at ratios of 3:1, 1:1, and 1:3. The second- and third-generation constructs were able to target tumor cells with varying levels of GPC3 expression, revealing broad tumor-targeting potential. Example 9: Cytotoxicity Assay: Gastric Cancer

[0295] To evaluate the potential cytotoxic effects of second- or third-generation CAR-T-transduced T cells in the gastric cancer tumor setting, a non-radioactive CytoTox cytotoxicity assay (Promega) was performed. In short, anti-GPC3 CAR-T cells (92-28Z, 92-28BBZ, or empty vector) were co-cultured with KATO-III at effector:target ratios of 1:3, 1:1, and 3:1, at 10,000 cells / well, and incubated at 37°C for 18 hours. Results

[0296] Cytotoxicity data showed that the second and third generation constructs exhibited considerable cytotoxicity against the gastric cancer tumor line KATO-III (Figure 7B), while cells transduced with the empty vector showed no activity. The data indicate that CAR-T cells are specific to their tumor targets and do not show reactivity to tissues that do not express the GPC3 antigen. Instruction manual, pages 47 / 55, 50 CN 121102467 A Example 10: Comparison of scFv 33 and scFv 92

[0297] To compare CAR-T cells with different scFv values ​​against GPC3, second- or third-generation CAR-T transduced T cells were used in the CytoTox non-radioactive cytotoxicity assay (Promega). In short, anti-GPC3 CAR-T cells 33-28Z, 92-28Z, 92-BBZ, 92-28BBZ, or empty vector were co-cultured with Huh-7 cells or control cells A431 (GPC3 negative) at an effector:target ratio of 3:1, 1:1, or 1:3, with 10,000 target cells per well, and incubated at 37°C for 18 hours. After incubation, absorbance data at visible wavelengths were collected using a standard 96-well plate reader. Results

[0298] Cytotoxicity data showed that at effector:target ratios of 3:1, 1:1, and 1:3, the construct with scFv 92 exhibited increased cytotoxicity compared to the construct with scFv 33 (Figure 10B). Cytotoxicity was antigen-specific, as no cytotoxicity was detected when co-cultured with the GPC3-negative tumor cell line A431 (Figure 10A). Example 11: Comparison of Second-Generation and Third-Generation CAR-T

[0299] To compare second-generation and third-generation CAR-T, CAR-T-transduced T cells were used in the CytoTox non-radioactive cytotoxicity assay (Promega). In short, anti-GPC3 CAR-T cells (4-28Z, 4-28BBZ, 4-4-28BBZ, 4-14-28BBZ, 4-20-28BBZ, 4-35-28BBZ, 4-42-28BBZ) or cells transduced with an empty vector were co-cultured with Huh7 (HCC) cells at an effector:target ratio of 3:1 or 1:1, with 10,000 target cells per well, and incubated at 37°C for 18 hours. After incubation, visible wavelength absorbance data were collected using a standard 96-well plate reader. Results

[0300] Cytotoxicity data showed that the second and third generation constructs were cytotoxic, with 4-20-28BBZ, 4-35-28BBZ, and 4-42-28BBZ exhibiting higher cytotoxicity, as measured by lysis increase (Figure 9). Example 12: Proliferation Assay

[0301] Proliferation of anti-GPC3 CAR-T cells after exposure to target cells (Huh-7, GPC3+) or control cells (SK-HEP-1, GPC3-) was determined by carboxyfluorescein diacetate dilution assay.

[0302] One week after transduction, control T lymphocytes and anti-GPC3 CAR-T cells were labeled with 1.5 μmol / L carboxyfluorescein diacetate diacetate succinimide (CFSE; Invitrogen) and plated with irradiated tumor targets (GPC3-positive and GPC3-negative lines) at an effector:target (E:T) ratio of 5:1. CFSE dilution on CD4+ and CD8+ T cells was measured by flow cytometry on day 4 of co-culture. Example 13: Tumor graft mouse model and lymphopenia from patients

[0303] NOD / SCID mice were each implanted with a 2x2x2 mm lung cancer tumor. Approximately day 27 post-implantation or when the tumor size reachedAt a tumor size of 220 mm³, mice were intravenously administered cyclophosphamide and intraperitoneally administered 1 x 10⁷ empty vector, hu92-28Z, or hu92-28BBZ CAR-T cells. A second administration was given on day 34. Tumor volume was measured using calipers approximately every 3–7 days until day 52 post-inoculation. Results

[0304] On day 51 post-inoculation, mice treated with second-generation CAR-T 92-28Z showed significantly smaller tumor size compared to mice treated with third-generation CAR-T 92-28BBZ or saline (Figures 8A and 8B). Mice treated with second-generation CAR-T 92-28Z also showed significantly reduced tumor weight compared to mice treated with third-generation CAR-T 92-28BBZ or saline (Figure 8C). Mice treated with second-generation CAR-T 92-28Z showed significantly reduced inhibition compared to mice treated with third-generation CAR-T 92-28BBZ or saline (Figure 8D). Example 14: Xenograft HCC Mouse Model

[0305] 0.5 x 10⁶ Huh-7 tumor cells were implanted into the left flank of 8- to 12-week-old NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ(NSG) mice (Bar Harbor, ME). Seven days after tumor inoculation, mice were treated with 1 x 10⁷ empty vector-transduced T cells, hu92-28Z, or hu92-28BBZ via tail vein injection. After treatment, mice were monitored at least weekly by caliper measurement for 35 days after tumor cell inoculation. Results

[0306] Compared with mice treated with T cells transduced with an empty vector, treatment with hu92-28Z and hu92-28BBZ showed significant antitumor activity. Figure 11A shows that mice treated with hu92-28Z or hu92-28BBZ had significantly reduced tumor volume on day 35 after tumor cell inoculation. Figure 11B shows that mice treated with hu92-28Z or hu92-28BBZ had significantly smaller tumors compared with mice treated with cells transduced with an empty vector, as demonstrated by the reduction in their weight. Figure 11C shows an image of the xenograft tumor. Example 15: Clinical Expansion of Anti-GPC3 CAR T Cells

[0307] To generate a large number of transduced T cells, a rapid expansion protocol (REP) was used to induce cell proliferation. Before being used for REP, the T cells were cultured with anti-CD3, anti-CD28, and IL-2 and transduced on the second day after the culture initiation as described above. Cells were cultured in 75 cm² flasks at 37°C and 5% CO₂. Cell counts were performed every two days, and cells were resuspended at a concentration of 0.5 × 10⁶ cells / mL in a solution containing 300 IU / mL.In fresh T-cell culture medium containing IL-2, the remaining time was spent holding them in culture. Example 16: Clinical Trial:

[0308] Patients with evaluable hepatocellular carcinoma underwent apheresis to isolate peripheral blood mononuclear cells. Lymphocytes were isolated, transduced with anti-GPC3 CAR virus, amplified, and aliquoted for immunological testing. On days -7 and -6 prior to CAR-T administration, patients underwent a 1-hour intravenous cyclophosphamide pretreatment regimen of 60 mg / kg / day x 2 days. On days -7 and -3 prior to CAR-T administration, patients underwent a 30-minute intravenous infusion (IVPB) of fludarabine for 5 days. During the pretreatment regimen, patients underwent daily complete blood count (CBC) testing.

[0309] In the first part of the Phase I study, one patient in each group started dose escalation with an initial dose of 109 anti-GPC3 CAR-T cells per patient. Individual patients were treated with a half-logarithmic increment. Therefore, the following doses will be used: 10⁹ cells, 3 x 10⁹ cells, 10¹⁰ cells, 3 x 10¹⁰ cells, and up to 1 x 10¹¹ cells. Autologous anti-GPC3 CAR-T cells will be administered intravenously over 20 to 30 minutes via a non-filtering tube.

[0310] All patients will return to the clinic for evaluation 6 weeks after administration of the CAR-T cell product.

[0311] Table 12: Sequence Specification Pages 49 / 55 52 CN 121102467 A Specification Pages 50 / 55 53 CN 121102467 A Specification Pages 51 / 55 54 CN 121102467 A Specification Pages 52 / 55 55 CN 121102467 A Specification Pages 53 / 55 56 CN 121102467 A Specification Pages 54 / 55 57 CN 121102467 A Specification Pages 55 / 55 58 CN 121102467 A Figure 1 Specification Drawing 1 / 16 Pages 59 CN 121102467 A Figure 2 Specification Drawing 2 / 16 Pages 60 CN 121102467 A Figure 3 Figure 4A Specification Drawing 3 / 16 Pages 61 CN 121102467 A Figure 4B Figure 5A Instruction manual, Figure 5B, page 4 / 16, CN 121102467 A; Figure 6A, page 6 / 16, CN 121102467 A; Figure 6B, page 6 / 16, CN 121102467 A; Figure 6C, page 7 / 16, CN 121102467 A.Figure 7A on page 8 / 16 of the specification; Figure 7B on page 9 / 16 of the specification; Figure 8A on page 10 / 16 of the specification; Figure 8A on page 11 / 16 of the specification; Figure 8B on page 12 / 16 of the specification; Figure 8D on page 12 / 16 of the specification; Figure 10A on page 13 / 16 of the specification; Figure 10A on page 14 / 16 of the specification; Figure 11A on page 15 / 16 of the specification; Figure 11C on page 16 / 16 of the specification; Figure 74 on page 121102467 A. Abstract A method for treating a subject expressing a solid tumor of GPC3 comprising administering to the subject an anti-GPC3 chimeric antigen receptor immune response cell, wherein the administration is performed after or simultaneously with subjecting the subject to lymphocyte reduction treatment. A kit for use in the method is also provided.

Claims

1. A method for treating a subject with a solid tumor expressing phosphatidylinositol proteoglycan-3 (GPC3), the method comprising administering anti-GPC3 chimeric antigen receptor immune response cells to the subject, wherein the administration is performed after or concurrently with lymphopenia treatment received by the subject, wherein the lymphopenia treatment comprises administering cyclophosphamide and fludarabine to the subject.

2. The method according to claim 1, wherein the immune response cells are NK cells (anti-GPC3-CAR NK cells) or T cells (anti-GPC3-CAR T cells).

3. The method of claim 2, wherein the administration of the anti-GPC3-CAR T cells to the subject is performed after the subject has received lymphopenia treatment.

4. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is at least about 5 × 10⁻⁶. 4 per kg.

5. The method of claim 2, wherein the amount of anti-GPC3-CAR T cells administered to the subject is approximately 5 × 10⁻⁶. 4 To approximately 1×10 12 per kg.

6. The method of claim 1, wherein the application effectively reduces the tumor size by at least 30%, the tumor size being measured by computed tomography (CT) scan.

7. The method of claim 1, wherein the application effectively stabilizes tumor size, wherein the stabilized tumor size is a baseline measurement of the tumor lesion diameter as measured by computed tomography (CT) scan with a change of less than 10%.

8. The method of claim 2, wherein the combination of administration of anti-GPC3-CAR T cells and lymphopenia treatment synergistically extends the median survival of the subject by at least about 6 months compared to administration of the anti-GPC3-CAR T cells alone.

9. The method according to claim 1, wherein the solid tumor is liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, cervical cancer, pancreatic cancer, liposarcoma, testicular non-seminomatous germ cell cancer, melanoma, adrenal adenoma, schwannoma, malignant fibrous histiocytoma, or esophageal cancer.

10. The method of claim 1, wherein the anti-GPC3 chimeric antigen receptor comprises an antigen-binding unit that specifically binds to the C-terminus of GPC3.

11. The method of claim 10, wherein the antigen-binding unit comprises a sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

12. The method of claim 1, wherein the anti-GPC3-CAR comprises a sequence selected from SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 or SEQ ID NO:

30.

13. The method of claim 1, wherein the anti-GPC3-CAR comprises a sequence selected from SEQ ID NO 28, SEQ ID NO 29 or SEQ ID NO 30.

14. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least two intracellular signal transduction domains.

15. The method of claim 2, wherein the anti-GPC3-CAR T cell comprises at least three intracellular signal transduction domains.

16. The method of claim 14, wherein the intracellular signal transduction domain is selected from the signal transduction domains of CD3, CD28, 4-1BB, OX40, DAP10, or ICOS.

17. The method of claim 1, wherein the lymphopenia treatment comprises reducing the amount of regulatory T cells in the subject.

18. The method of claim 17, wherein reducing the amount of regulatory T cells comprises reducing the number of said regulatory T cells by at least about 30%, the amount of said regulatory T cells being determined by the amount of circulating CD4+ in the subject. + and CD25 + Flow cytometry analysis of cells.

19. The method of claim 1, wherein the lymphopenia treatment further comprises administering radiation or a biological agent to the subject.

20. The method of claim 1, wherein the lymphopenia treatment further comprises administering other chemotherapy to the subject.

21. The method of claim 20, wherein administering other chemotherapy to the subject comprises administering a chemotherapeutic agent selected from etoposide, cytarabine, methotrexate, vincristine, doxorubicin, and any combination thereof.

22. The method of claim 21, wherein the chemotherapeutic agent is administered to the subject at least once prior to the administration of the anti-GPC3-CAR T cells.

23. The method of claim 1, wherein the lymphopenia treatment reduces the number of lymphocytes by at least about 20%, the number of lymphocytes being measured by complete blood count (CBC) analysis.

24. The method of claim 2, further comprising administering the anti-GPC3-CART cells to the subject a second time.

25. The method of claim 1, wherein the subject suffers from a refractory, persistent, or progressive disease.

26. The method of claim 2, wherein the anti-GPC3-CAR T cells are autologous or allogeneic relative to the subject.

27. The method of claim 1, further comprising administering at least one immunostimulant to the subject concurrently with or after the administration of the anti-GPC3 chimeric antigen receptor immune response cells.

28. The method of claim 27, wherein the immunostimulant is selected from interleukin-2 (IL-2), IL-3, IL-6, IL-11, GM-CSF, and any combination thereof.

29. The method of claim 19, wherein the biological agent is an antibody that targets an antigen expressed on lymphocytes.

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